Ezrin assay method for the in vitro diagnosis of colorectal cancer

ABSTRACT

A method for the in vitro diagnosis of colorectal cancer by determining the presence of the Ezrin tumor marker in a biological sample taken from a patient suspected of having colorectal cancer using at least one anti-Ezrin monoclonal antibody directed against an Ezrin epitope chosen from the epitopes of sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4+SEQ ID No. 5, SEQ ID No. 6+SEQ ID No. 7 and SEQ ID No. 8. Said method can be used for early diagnosis, screening, therapeutic follow-up and prognosis, and also for relapse diagnosis in relation to colorectal cancer.

The present invention relates to the cancerology field. Moreparticularly, the subject of the present invention is a method for thein vitro diagnosis of colorectal cancer in a human patient, bydetermining the presence of Ezrin in a biological sample taken from thispatient, it being possible for said method to be used both for earlydiagnosis, screening, therapeutic follow-up and prognosis, and forrelapse diagnosis in relation to colorectal cancer.

Colorectal cancer (CRC) is a major public health problem. The worldwideincidence thereof was estimated at 875 000 new cases in 1996¹. Takinginto account both sexes, it is the cancer that occurs most frequently inwestern countries, where it is generally classed among the first 3 mostcommon causes of death due to cancer. The 5-year survival rate, allstages taken into account, is in the region of 60%.

Only early diagnosis offers the hope of a curative treatment. However,at the current time, there is no serological screening test nor specificdiagnostic test which is early.

Screening for colorectal cancer is currently carried out in Europe withtwo distinct approaches: firstly, using a paraclinical test whichconsists in looking for the presence of blood in the stools (FaecalOccult Blood Test, FOBT, marketed, for example, under the nameHemoccult®). This technique has demonstrated its clinical usefulness.When it is used every 2 years in individuals between the ages of 50 and74, it can reduce by 15 to 20% mortality due to colorectal cancer². Forthis, it is necessary for more than half the population concerned toparticipate regularly in the screening and for a colonoscopy to becarried out in the event of a positive test, optionally followed by anappropriate treatment.

Nevertheless, this screening technique suffers from a certain number ofhandicaps:

The major drawback of this test is its mediocre sensitivity, mostespecially for adenomas (precancerous dysplastic lesion) which, if theyare large in size, will result in the development of cancer in 1 caseout of 10.

The test is also not very specific. The appearance of blood in thestools may be related to a nontumor condition: ulcerative colitis,hemorrhoids, fistulae, etc. In this case, an investigation bycolonoscopy must be carried out, with the drawbacks describedhereinafter.

Finally, Hemoccult® tests are difficult to interpret; they musttherefore be read in specialized centers, by qualified competentpersonnel.

Immunological tests specific for human hemoglobin (Feca EIA®, HemeSelect®, etc.) have also been described. They probably constituteprogress compared with Hemoccult®, but they essentially exhibit the sameproblems. Thus, InSure™, marketed by Enterix Inc., makes it possible todetect 87% of patients suffering from CRC and 47% of those havingprecancerous polyps. It is a test for detecting human hemoglobin in thestools, and more particularly the globin portion of this molecule.

A second screening strategy is the systemic performing of a colonoscopyafter the age of 50, which makes it possible in theory to reducemortality due to colorectal cancer. However, the acceptability of thisexamination in individuals who are in good health is too low for ascreening policy using endoscopy to reduce mortality (the level ofcompliancy for colonoscopy in European countries having set up thisscreening strategy is around 2%). There is a not insignificant risk(0.1%) of perforation and bleeding of the colon and of death ( 1/10000), and it is also very expensive for public health. Furthermore,colonoscopy requires a very restrictive prior colonic preparation, whichin large part explains the poor compliance.

Tumor markers that can be assayed by immunoassays have for a long timebeen described in the context of colorectal cancer. They are inparticular the carcinoembryonic antigen (CEA) and CA19-9.

CEA is used for follow-up. It cannot be used for the screening or forthe early diagnosis of colorectal cancer because its sensitivity and itsspecificity are insufficient. This is because this marker is expressedby other types of cancer, and in benign pathologies. Despite everything,it is possible to increase sensitivity without losing specificity bycombining, with CEA, another tumor marker such as CA19-9 or CA72-4.

The causes of physiological variations in CA19-9 are rare, but otherbenign conditions (hepatobiliary conditions, pancreatic conditions), ormalignant conditions may induce an increase in CA19-9. This marker,taken alone, is therefore also of no interest for diagnosis.Nevertheless, since its serum concentration is correlated with the sizeof the tumor and the presence of metastases, it may also enable atherapeutic follow-up or the early demonstration of relapses.

Commercially available tests have, moreover, been proposed, such as:

-   -   Colopath®/ColorectAlert^(MD), marketed by Ambrilia, is a rapid        and relatively noninvasive screening test for CRC. Colopath®        detects a plasmalogen (class of complex lipids which are part of        phospholipids) in the rectal mucus of individuals with a        colorectal pathological condition, whereas ColorectAlert^(MD)        detects T-antigen, a complex sugar in the rectal mucus. The        Colopath®/ColorectAlert^(MD) test involves the application of        rectal mucus to a test strip, and the positive or negative        result is based on a Schiff reaction. Ambrilia has studied 1787        individuals and demonstrated that Colopath®/ColorectAlert^(MD)        detects 54% of cases of early-stage colorectal cancer and 49% of        all stages combined.    -   COLARIS, marketed by Myriad Genetics, is a test for detecting,        in the blood, mutations in the MLH1 and MSH2 genes for screening        for hereditary nonpolyposis colon cancer (HNPCC syndrome). The        result of the test is available in 3 weeks. Myriad uses the most        sensitive and most specific sequencing techniques currently        available. The test is expensive.    -   DR-70®, marketed by AMDL, is a test to screen for various types        of cancer (lung, colon, breast, liver, stomach, etc.). It is not        therefore specific for CRC. The principle of said test is based        on the double sandwich ELISA technique (assaying of the DR-70        antigen). Revealing is carried out by enzymatic reaction        (antibodies coupled to biotin and to streptavidin). A colored        reaction indicates the presence of cancer.

The Ezrin marker (Swiss Prot No. P15311, also known as p81, Cytovillinor Villin-2) is a protein which provides binding between the cellmembrane and the Actin filaments of the cytoskeleton of the cell, inparticular in the microvilli of intestinal epithelial cells³. W. G.Jiang and S. Hiscox⁴ have shown that the Interleukins IL-2, IL-8, IL-10,etc. can inhibit the expression of ezrin in the HT29 human colorectalcancer cell line. The same authors⁵ have shown that the inhibition ofEzrin expression in the HT115 and HRT18 colorectal cancer cell linesreduces the adhesion between cells and increases the mobility and theinvasive behavior of the cells. They have concluded that Ezrin regulatescell/cell and cell/matrix adhesions by interacting with the celladhesion molecules E-Cadherin and beta-Catenin. They have suggested thatEzrin could play an important role in controlling the invasive potentialof cancer cells. Moreover, T. Xiao et al.⁶ have used an ELISA assay toquantify the plasma Ezrin of patients with lung cancer. However, theyhave not observed any differences compared with control individuals.Finally, in patent application WO 2006/015079, Ezrin has been describedas a diagnostic marker for certain cancers, such as colon cancer.However, only ovarian cancer and endometrial cancer have beenillustrated, such that, to date, no sensitive and specific diagnosticmethod for the diagnosis of colorectal cancer using Ezrin as a marker isavailable.

The applicants have now demonstrated, surprisingly, that colonic tumorsnot only specifically secrete Ezrin, but especially release it out ofthe cancerous tissue, as will be demonstrated in greater detailhereinafter, that its concentration in the biological sample in whichthe method of the invention is implemented is increased compared withthe reference values determined for healthy patients, and that the useof particular anti-Ezrin monoclonal antibodies makes it possible toprovide a sensitive and specific method for the diagnosis of colorectalcancer.

Thus, a first subject of the present invention is a method for the invitro diagnosis of colorectal cancer by determining the presence ofEzrin in biological samples taken from patients suspected of havingcolorectal cancer, and preferably remote from the tumors, using at leastone anti-Ezrin monoclonal antibody directed against an Ezrin epitopechosen from the epitopes of sequence SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 4+SEQ ID No. 5, SEQ ID No. 6+SEQ ID No. 7 and SEQ IDNo. 8.

The present invention also relates to the use of this method both forearly diagnosis, screening, therapeutic follow-up and prognosis, and forrelapse diagnosis in relation to colorectal cancer.

The method of the invention therefore makes it possible to diagnosecolorectal cancer specifically and early by means of a simple testconsisting in searching for the presence of Ezrin in a biological sampletaken from a patient, which is preferably remote from the potentialtumor.

The determination, in a biological sample which may or may not be remotefrom the tumor, of the presence of Ezrin then makes it possible toconclude with respect to the pathological condition sought. One of theadvantages of the method of the invention also lies in the possibilityof using a sample remote from the potential tumor as a diagnosticsample, thereby enabling a simple and noninvasive diagnosis, whereas atissue diagnosis requires a biopsy taken invasively. In fact, the studyof tissue markers, for example on a tissue section(immunohistochemistry), may be of prognostic interest, but is of nointerest for screening for or diagnosing colorectal cancer.

The expression “determining the presence of a tumor marker” is intendedto mean determining the presence of the protein using at least oneanti-Ezrin monoclonal antibody directed against an Ezrin epitope chosenfrom the epitopes of sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,SEQ ID No. 4+SEQ ID No. 5, SEQ ID No. 6+SEQ ID No. 7 and SEQ ID No. 8.

The term “epitope” is intended to mean a peptide having at least thesequences as defined by the sequences SEQ ID No. 1 to 8, and at most 10,8, 6 or 4 additional amino acids distributed on either side of thesequence under consideration, homogeneously or nonhomogeneously, or elseon just one side, and also the analogs and homologs thereof.

In general, the term “analog” refers to peptides having a sequence and anative polypeptide structure exhibiting one or more amino acidadditions, substitutions (generally conservative in terms of nature)and/or deletions compared with the native molecule, insofar as themodifications do not destroy the antigenic reactivity.

The analogs that are particularly preferred include substitutions whichare conservative in nature, i.e. substitutions which take place in anamino acid family. Specifically, amino acids are generally divided upinto 4 families, namely (1) acidic amino acids such as aspartate andglutamate, (2) basic amino acids such as lysine, arginine and histidine,(3) nonpolar amino acids such as alanine, leucine, isoleucine, proline,phenylalanine, methionine and tryptophan, and (4) uncharged polar aminoacids such as glycine, asparagine, glutamine, cysteine, serine,threonine and tyrosine. Phenylalanine, tryptophan and tyrosine aresometimes classed as aromatic amino acids. For example, it can bereasonably predicted that an isolated replacement of leucine withisoleucine or with valine, of an aspartate with a glutamate or of athreonine with a serine, or a similar conservative replacement of oneamino acid with another amino acid that is structurally related, willhave no major effect on the biological activity. Those skilled in theart will readily determine the regions of the peptide molecule ofinterest that can tolerate a change with reference to Hopp/Woods andKyte-Doolittle plots, well known in the art.

The term “homology” is intended to mean the percentage identity betweentwo peptide molecules. Two amino acid sequences are “substantiallyhomologous” to one another when the sequences exhibit at least 60%,preferably at least 75%, more preferably at least 80-85%, morepreferably at least 90% and more preferably at least 95-98% or moresequence identity over a defined length of the peptide molecules.

The expression “epitope of sequence SEQ ID No. X+SEQ ID No. Y” isintended to mean an epitope constituted of two distinct parts of theprotein from which the epitope is derived, the antibody which recognizesthis epitope having to recognize these two parts. This recognition isvery probably linked to the sequences being in proximity in thethree-dimensional structure of the protein.

The expression “release by colonic tumors” is intended to mean theactive or passive secretion or the release, whatever the mechanism, ofthe tumor marker by the tumor cells themselves or by the neighboringnontumor cells following lesions or modifications of cell phenotyperesulting from the tumor development.

The expression “biological sample in which the method of the inventionis carried out” is intended to mean any biological sample capable ofcontaining the tumor marker of interest. By way of example of abiological sample not remote from the tumor, mention may be made ofsolid samples such as the tissue originating from the tumor, frombiopsies of this tumor, from lymph nodes or from metastases of thepatient, and cells purified from these solid samples. By way of exampleof a biological sample remote from the tumor, mention may be made ofbiological fluids such as whole blood or derivatives thereof, forexample serum or plasma, urine, saliva and effusions, bone marrow andstools, and cells purified from these liquid samples. Blood orderivatives thereof and also stools, effusions and cells purified fromthese liquid samples are preferred.

The method of the invention may be improved by detecting, in addition toEzrin, at least one other tumor marker, where appropriate also releasedby colonic tumors out of the cancerous tissues. Thus, the combination ofat least two markers makes it possible to improve the specificity andthe sensitivity of the test for the diagnosis of colorectal cancer.

Thus, another subject of the invention also consists in determining thepresence of at least one other tumor marker chosen from the followingtwo groups of markers, considered alone or in combination:

-   -   group A: Leucocyte Elastase Inhibitor, Aminoacylase 1, Liver        Fatty Acid-Binding Protein, Intestinal Fatty Acid-Binding        Protein, Apolipoprotein AI, Apolipoprotein AII and I-Plastin,        some of these markers being new markers identified by the        applicant,    -   group B: markers having an additional diagnostic interest,        namely: Beta2-Microglobulin, Proteasome 20S, Galectin-3,        L-Lactate Dehydrogenase Chain B, Calreticulin, Regenerating        Islet-Derived Protein 3 Alpha, Tumor-Associated Calcium Signal        Transducer 1, Keratin type II Cytoskeletal 8, Keratin type I        Cytoskeletal 18, Keratin type I Cytoskeletal 19, Epithelial        Cadherin, CEA, Villin, CA19-9, CA 242, CA 50, CA 72-2,        Testosterone, TIMP-1, Cripto-1, Intelectin-1, Protein Disulfide        Isomerase, Cytokeratin 20, Translationally-Controlled Tumor        Protein, (Pro)defensin-A5, the detection of DNA fragments in the        blood having specific alterations to their methylation profile,        for instance methylated DNA of the AXL4 gene (aristaless-like        homeobox-4 gene methylation) or the methylated DNA of the        septin-9 gene, the detection of specific alterations in fecal        DNA fragments, such as specific mutations of fecal DNA or        specific alterations of the methylation profile of fecal DNA,        the detection of human fecal hemoglobin.

The method of the invention may therefore be improved by detecting atleast two markers, one being Ezrin, the other being another tumor markerchosen from group A, namely, Leucocyte Elastase Inhibitor, Aminoacylase1, Liver Fatty Acid-Binding Protein, Intestinal Fatty Acid-BindingProtein, Apolipoprotein AI, Apolipoprotein AII and I-Plastin.

The “newly described tumor marker” is intended to mean the protein orthe messenger RNA or specific modifications of the corresponding gene,such as mutations or methylations.

The Leukocyte Elastase Inhibitor tumor marker (Swiss Prot No. P30740,also known as LEI, Serpin B1, Monocyte/neutrophil elastase inhibitor,M/NEI or EI) was sequenced in 1992⁷. LEI specifically inhibits proteaseshaving elastase-type or chymotripsin-type properties by formation of acomplex that cannot be dissociated under the action of SDS⁸. LEI thusinhibits three of the major proteases produced by neutrophils: LeukocyteElastase, Proteinase-3 and Cathepsin G. These proteases enable theimmune system to defend the organism by proteolysis of extracellular orphagocytosed substrates. However, when these proteases are in excess,they are responsible for inflammatory reactions. LEI could thereforehave a role in regulating and limiting the inflammatory action inducedby cell proteases. The applicant has shown, for its part, surprisingly,that this protein is a good marker in biological samples taken from apatient having colorectal cancer, said samples being remote or not fromthe tumor.

The Aminoacylase 1 marker (Swiss Prot No. Q03154, also known as EC3.5.1.14, N-Acyl-L-Amino Acid Amidohydrolase or ACY-1) is part of theAminoacylase family. They are enzymes which catalyze the hydrolysis ofacylated amino acids so as to give fatty acids and amino acids⁹. Animmunochemical assay for Aminoacylase enzymatic activity was developedas early as 1975 by K. Lorentz et al.¹⁰ and was used to assay varioustissues and sera¹¹. The study showed an increase in Aminoacylaseactivity in the case of hepatic pathological conditions but not in thecase of colon cancer. Moreover, the Aminoacylase 1 gene has beenidentified on chromosome 3p21.1¹². The 3p21.1 region is reduced tohomozygosity in a small cell lung cancer, and in this case, theAminoacylase expression is repressed or undetectable¹³. Similarly, S.Balabanov et al.¹⁴ have shown that the Aminoacylase expression isrepressed in the case of kidney cancer. The applicant has shown, for itspart, surprisingly, that this protein is a good marker in biologicalsamples taken from a patient having colorectal cancer, said samplesbeing remote or not from the tumor.

The Liver Fatty Acid-Binding Protein marker (Swiss Prot No. P07148, alsoknown as L-FABP, FABP1, FABPL, Z-protein or sterol transporter protein)belongs to the FABP family which comprises nine isoforms. Each isoformis named according to the tissue in which it was first detected. Theseisoforms have a shared function and similar three-dimensionalstructures, but their sequence homology is not high. L-FABP wassequenced in 1985¹⁵. It is a small protein of 15 kDa that is abundant inthe cytosol and that has the ability to bind to free fatty acids andalso to bilirubin. Some recent studies appear to indicate thatimpairments in expression of the L-FABP protein could induce atumorigenesis process. For prostate cancer, the level of expression ofL-FABP mRNAs in tumor tissue biopsies was 10 times higher than in thenormal tissue¹⁶. For colon cancer, several teams have identified adecrease in the expression of L-FABP protein in the tumor tissuecompared with normal colonic mucosa, using 2-dimensional electrophoresistechniques¹⁷. This result has also been confirmed byimmunohistochemistry techniques. In addition, the L-FABP protein is aprognostic liver resection marker in patients with colorectal cancerhaving metastasized to the liver¹⁸. The applicant has shown, for itspart, surprisingly, that this protein is a good marker in biologicalsamples taken from a patient having colorectal cancer, said samplesbeing remote from the tumor.

The Intestinal Fatty Acid-Binding Protein marker (Swiss Prot No. P12104,also known as I-FABP, FABP-2 or FABPI) was sequenced in 1987¹⁹. It is asmall protein of 15 kDa that is abundant in the cytosol and that has theability to bind to free fatty acids and also to bilirubin. The I-FABPprotein is expressed in the enterocytes of the small intestine and mayconstitute approximately 2% of the protein content of this cell type. Atthe tissue level, the duodenum and the jejunum contain significantlyhigher amounts of I-FABP than the colon (jejunum: 4.8 μg/g, colon: 0.25μg/g)²⁰. I-FABP could not be detected in the plasma samples of healthyindividuals. On the other hand, in certain pathological contexts such asintestinal ischemia, Crohn's disease or primary biliary cirrhosis, it ispossible to demonstrate an increase in the plasma I-FABP concentrationin certain individuals²⁰. For prostate cancer, it has been shown thatthe level of expression of I-FABP mRNA in biopsies of tumor tissue is 7times higher than in normal tissue¹⁶. In the model of induction of acolorectal tumor with azoxymethane in the rat, the level of expressionof I-FABP mRNA is reduced by 2.92 to 3.97 times when the animals have adiet that reduces the incidence of cancer (soya proteins or wheyhydrolysate)²¹. The applicant has shown, for its part, surprisingly,that this protein is a good marker in biological samples taken from apatient having colorectal cancer, said samples being remote from thetumor.

The Apolipoproteins are a family of proteins constituted of polar aminoacids, which allow the transport of lipids in the blood by formation ofa hydrophilic macromolecular complex called a lipoprotein. For each ofthe human plasma Apolipoproteins, there are isoforms derived fromgenetic polymorphism and/or from post-translational modifications, thepresence of which in the blood can be associated with certainpathological conditions²². The plasma concentration of Apolipoproteinsis not insignificant, of the order of those mg/ml²³.

The Apolipoprotein AI marker (NCBI No. 490098, also known as Apo A-I,Apo AI and Apo A1) is a protein of 243 amino acids and of 28 kDa. It isessentially synthesized by the liver and the intestine. This protein hasbeen shown to be underabundant in the sera of patients suffering fromcolorectal cancer compared with healthy individuals, by SELDI-TOF²⁴.However, it is specified in this article that patients with CRC aredistinguished from healthy individuals by combining Apo AI with otherprotein markers. Moreover, this article specifies that the assaying ofApo AI by turbidimetric immunoassay, carried out by another team, doesnot confirm the underabundance of this protein in the sera of patientshaving CRC²⁵. Hachem et al.²⁶ have, for their part, assayed Apo AI insera of patients having had liver cancer following colorectal cancermetastases. The applicant has shown, for its part, surprisingly, thatassaying by immunoassay makes it possible to demonstrate a decrease inthe concentration of this protein in patients having colorectal cancer,contrary to what was put forward by Engwegen et al.²⁴, who were able todemonstrate this decrease only by implementing the SELDI-TOF technique.The assaying of Apo AI by immunoassay in biological samples is a goodmethod for the diagnosis of colorectal cancer, said samples being remotefrom the tumor, insofar as the assaying by immunoassay that is carriedout is not turbidimetry as used by the team of Zhang et al.²⁵.

The Apolipoprotein AII marker (Swiss Prot No. P02652, also known as ApoAII, Apo-AII and Apo A2) is a 17380-Da protein composed of twopolypeptide chains of 77 amino acids each, linked by a disulfide bridge.Like Apolipoprotein AI, Apolipoprotein AII is essentially synthesized bythe liver and the intestine. Hachem et al.²⁶ have also assayed, inaddition to Apo AI, Apo AII in sera of patients having had liver cancerfollowing colorectal cancer metastases. However, the results are notsignificant and do not enable a conclusion to be drawn as to thepathological condition sought. The applicant has shown, for its part,surprisingly, that the decrease in the concentration of this protein inpatients having colorectal cancer makes it a good marker in biologicalsamples taken from a patient having colorectal cancer, said samplesbeing remote from the tumor.

The I-Plastin marker (Swiss Prot No. Q14651, also known asintestine-specific Plastin or Plastin 1) belongs to the family of humanPlastins of which three representatives are known: I-Plastin, L-Plastinand T-Plastin. Some authors call Plastins “Fimbrins”, yet other authorsreserve the name Fimbrin for I-Plastin. The Plastins are proteins thatbind to Actin so as to form the cytoskeleton (cell skeleton). They are70 kDa proteins that are relatively well-conserved throughout eukaryoticevolution. They exhibit strong tissue specificity, only one isoform at atime is present in normal tissues²⁷. The use of Plastins with respect tocancer has already been described in U.S. Pat. No. 5,360,715, whichproposes a method for determining whether a cell is hematopoietic orneoplastic i.e. cancerous. This method claims the assaying of L-Plastinand of T-Plastin at the cellular level, and more particularly theassaying of the mRNA thereof. However, despite these properties, noprior study has been carried out to evaluate the importance of Plastinsin relation to the diagnosis of colorectal cancer using a serum or stoolsample. Furthermore, I-Plastin has never even been envisioned as apotential cancer marker²⁸. The applicant has shown, for its part,surprisingly, that this protein is a good marker in biological samplestaken from a patient having colorectal cancer, said samples being remoteor not from the tumor.

The concentration of the tumor marker chosen from group A will,depending on the marker under consideration, be increased or decreasedin the biological sample in which the method of the invention is carriedout, compared with the reference values determined for the healthypatients.

The method of the invention may also be improved by combining thedetection of Ezrin and of one other tumor marker chosen from group B,namely: the markers Beta2-Microglobulin, Proteasome 20S, Galectin-3,L-Lactate Dehydrogenase Chain B, Calreticulin, RegeneratingIslet-Derived Protein 3 Alpha, Tumor-Associated Calcium SignalTransducer 1, Keratin type II Cytoskeletal 8, Keratin type ICytoskeletal 18, Keratin type I Cytoskeletal 19, Epithelial Cadherin,CEA, Villin, CA19-9, CA 242, CA 50, CA 72-2, Testosterone, TIMP-1,Cripto-1, Intelectin-1, Protein Disulfide Isomerase, Cytokeratin 20,Translationally Controlled Tumor Protein, (Pro)defensin-A5, thedetection of DNA fragments in the blood having specific alterations totheir methylation profile, for instance methylated DNA of the AXL4 gene(aristaless-like homeobox-4 gene methylation) or methylated DNA of theseptin-9 gene, the detection of specific alterations in fecal DNAfragments, such as specific mutations of fecal DNA or specificalterations of the methylation profile of fecal DNA, and the detectionof human fecal hemoglobin. Of course, the method of the invention mayalso implement the detection, in the same assay, of Ezrin, of at leastone tumor marker chosen from group B and of at least one other tumormarker chosen from group A.

The Beta2-Microglobulin marker (Swiss Prot No. P61769, also known asβ2-Microglobulin, β2M) is a low-molecular-weight (11 to 12 kDa) proteinfound at the surface of most nucleated human cells. The serumβ2-Microglobulin level increases in certain patients suffering fromcancer, without this increase being specific, or correlated with thenature of the tumor, its stage or the severity of the disease. Asignificant increase is also observed in other diseases, such as lupuserythematosus, rheumatoid arthritis, Sjögren's syndrome, malignantdiseases of the lymphoid system (multiple myeloma, B-cell lymphoma),certain viral diseases (hepatitis or AIDS), and in hemophiliac patients.Since β2-Microglobulin is filtered by the renal glomeruli and reabsorbedby the proximal convoluted tubules, its concentration in the blood maybe modified in the case of renal pathological conditions. The assayingof β2-Microglobulin is thus most commonly reserved for the diagnosis ofrenal pathological conditions, or for the follow-up of infection withthe acquired immunodeficiency virus. However, this marker is known as atumor marker, in particular for colon cancer.

The Proteasome 20S marker (also known as Prosome) is the centralstructure of the proteasome, which is itself a molecular complexresponsible for the intracellular degradation of ubiquitinatedproteins²⁹. The Proteasome is a molecular complex of 700 kDa,constituted of 28 subunits associated in 4 rings of 7 subunits. Inhumans, 7 alpha units (α1, α2, α3, α4, α5, α6 and α7) and 10 beta units(β1, β2, β3, β4, β5, β6, β7, β1i, β2i and β5i) are known. By virtue ofits catalytic properties, the Proteasome plays a central role in themechanisms of cell proliferation, growth, regulation and apoptosis, andtherefore in the cancerization pathways. Proteasome inhibition withBortezomib (Velcade) is a recognized treatment for multiple myeloma.Phase II or III therapeutic trials are ongoing for hematological cancersor tumors. T. Lavabre-Bertrand et al.³⁰ have shown that the serum levelof Proteasome can increase on the occasion of certain pathologicalconditions, in particular in the case of cancers (myeloma, lymphoma andsolid tumors).

The Galectin-3 marker (Swiss Prot No. P17931, also known as Gal-3,Galactose-Specific Lectin 3, MAC-2 antigen, IgE-Binding Protein, 35 kDaLectin, Carbohydrate Binding Protein 35, CBP 35, Laminin-BindingProtein, Lectin L-29, L-31, Galactoside-Binding Protein or GALBP) is alectin capable of binding to beta-galactoside structures ofN-acetyllactosamine type. It is a protein with multiple functionsinvolved in various biological functions, including the adhesion oftumor cells, proliferation, differentiation, angiogenesis, apoptosis,metastatic cancer progression³¹. Various studies have shown that Gal-3can form complexes with numerous molecules: CEA, IgE, Laminin, Mucin,Mac-2BP, LAMP1, LAMP2, Fibronectin, etc. A serum assay of Gal-3 has beendescribed by I. Iurisci et al.³². Gal-3 was captured on microplatescoated with Mac-2-binding protein (a Gal-3-binding protein) and thenrevealed with an anti-Gal-3 rat antibody. This study showed elevatedserum of Gal-3 in the case of gastrointestinal cancers, breast cancer,lung cancer, ovarian cancer, melanomas and non-Hodgkin lymphomas.

The L-Lactate Dehydrogenase Chain B marker (Swiss Prot No. P07195, alsoknown as LDH-B, LDH Heart Unit or LDH-H) is a protein that can formcomplexes in the form of homotetramers. This protein can also formcomplexes with the L-Lactate Dehydrogenase Chain A protein (Swiss ProtNo. P00338, also known as LDH-A, LDH muscle unit or LDH-M) in the formof heterotetramers. The serum dose and/or the serum enzymatic activityof the tetrameric complexes, called LDH, increase(s) in the bloodstreamproportionally to the tumor mass for many solid tumors. Its use isrecommended in combination with human chorionic gonadotrophin (beta-hCG)and placental alkaline phosphatase for the follow-up of seminal vesiclecancers. LDH is considered to be a marker of importance for theprognosis of lymphomas, of leukemia and of colon cancer³³.

The Calreticulin marker (Swiss Prot No. P27797, also known as CRP55,Calregulin, HACBP, ERp60 or grp60) is a multifunctional protein. It is alectin capable of interacting transiently with virtually all themonoglycosylated proteins of the endoplasmic reticulum. D. J. McCool etal.³⁴ have thus shown that Calreticulin is involved in maturation of thecolonic mucin MUC2. A method for the diagnosis of CRC which usesassaying of Calreticulin in a tissue, the stools or a body fluid isdescribed in patent application WO 03/065003.

The Regenerating Islet-Derived Protein 3 Alpha marker (Swiss Prot No.Q06141, also known as Reg III-alpha, Pancreatitis-Associated Protein 1or Pancreatis Associated Protein I (PAP 1)) is a protein that is weaklyexpressed in the healthy pancreas. It is overexpressed during the acutephases of pancreatitis and in certain patients suffering from chronicpancreatitis. In this case, it appears in the pancreatic fluid and inthe bloodstream³⁵. Y. Motoo et al.³⁶ have shown, by ELISA assay, thatthe level of PAP 1 in the blood increases in certain patients havingcolon cancer, stomach cancer, liver cancer or pancreatic cancer, andalso in the case of renal insufficiency. To to this, they used the ELISAassay (PANCEPAP) marketed by the company Dynabio (La Gaude, France).

The Tumor-Associated Calcium Signal Transducer 1 marker (Swiss Prot No.P16422, also known as Major gastrointestinal tumor-associated proteinGA733-2, Epithelial cell surface antigen, EpCAM, Epithelialglycoprotein, EGP, Adenocarcinoma-associated antigen, KSA, KS 1/4antigen, Cell surface glycoprotein Trop-1 or CD326 antigen), wascharacterized in 1979 by virtue of its ability to be recognized by anantibody directed against colorectal cancer cells³⁷. This protein isknown by various names, as indicated above, but the most common use isto call it EpCAM. It is a transmembrane protein expressed at thebasolateral surface of cells, in certain epithelia and many cancers³⁸.As early as 1982, Herlyn et al.³⁹ showed that the injection of ananti-EpCAM monoclonal antibody could inhibit tumor growth in patientshaving colorectal cancer. These results resulted in the development ofan antitumor treatment based on an anti-EpCAM antibody calledEdrecolomab. This treatment is marketed under the name Panorex™.Moreover, H. Abe et al.⁴⁰ have shown, by ELISA assay, that a solubleform of EpCAM, called MK-1, is increased in the bloodstream in 10% ofcancer patients studied.

The Cytokeratins are part of the proteins that make up the intermediatefilaments of the cytoskeleton of epithelial cells. Currently, more than20 human Cytokeratins have been identified. The Cytokeratins 8 (SwissProt No. P05787, also known as Cytokeratin-8, CK-8, Keratin-8 or K8), 18(Swiss Prot No. P05783, also known as Cytokeratin-18, CK-18, Keratin-18or K18) and 19 (Swiss Prot No. P08727, also known as Cytokeratin-19,CK-19, Keratin-19 or K19) are the most abundant in epithelial cells andare useful tools for the diagnosis of cancer pathologies⁴¹. Thisclinical importance is linked to the release of Cytokeratins byepithelial cells in the apoptotic or proliferation phase. In the case ofapoptosis, this release occurs in the form of soluble fragments whichseem to appear under the proteolytic action of caspases. UndegradedCytokeratin forms have never been described in the bloodstream. Thethree Cytokeratin assays most commonly used clinically are the tissuepolypeptide antigen (TPA) assay, the tissue polypeptide specific antigen(TPS) assay and the CYFRA 21-1 assay. TPA is a broad-spectrum test whichmeasures Cytokeratins 8, 18 and 19. The TPS and CYFRA 21-1 assays aremore specific and measure, respectively, fragments of Cytokeratin 18 andof Cytokeratin 19. These 3 assays detect soluble Cytokeratin fragmentsthat may be present on their own or in the form of protein complexes.TPA, TPS or CYFRA-21-1 have been used for the therapeutic follow-up ofcolorectal cancers, breast cancers, lung cancers, bladder cancers,ovarian cancers, pancreatic cancers, prostate cancers and certain ENTcancers. The assaying of soluble Cytokeratin fragments in the blood infact has a clinical value in screening for relapses or evaluating theresponse to the therapy used (radiotherapy, chemotherapy, hormonetreatment). Regular assaying makes it possible in particular to evaluatethe progression of the tumor mass. The amount of soluble bloodCytokeratins also has a prognostic aspect with respect to the tumorstage and to the formation of metastases. Currently, the blood assay forCytokeratin that is most commonly used is CYFRA 21-1. It is highlyrecommended for the follow-up of patients having non-small cell lungcancer. Various commercially available assays exist for TPA (AB SangtecMedical Co., Byk-Roland, etc.), TPS (IDL Biotech AB, BEKI Diagnosiss,etc.) and CYFRA-21-1 (Roche Diagnosiss, CIS Bio-International, FujirebioDiagnosiss, etc.). Moreover, H. Kim et al.⁴² have shown that assayingfecal Cytokeratin 19 (DiNonA Inc.) may be useful in screening forgastrointestinal diseases, in combination with a fecal occult bloodassay. Finally, the use of Cytokeratin 20 (Swiss Prot No. P35900, alsoknown as Keratin, type I Cytoskeletal 20, CK-20, Keratin-20, K20, or ITprotein) as a marker in colorectal cancer is described in patentapplication US 2002/0160382.

The Epithelial Cadherin marker (Swiss Prot No. P12830, also known asE-Cadherin, Uvomorulin, Cadherin-1, CAM 120/80 or CD324 antigen) is atransmembrane protein that mediates calcium-dependent cell adhesion. Itis specifically expressed in epithelial cells, where it is involved inmaintaining their phenotype. The cytoplasmic domain of E-Cadherin bindsto β-Catenin, which is itself bound to the actin filament networks ofthe cytoskeleton. This E-Cadherin/β-Catenin binding plays an essentialrole in stabilizing cell/cell adhesions of the epithelial tissue. Theloss of E-Cadherin can therefore reduce cell adhesion and increase theinvasive capacity of cancer cells. A reduction in expression ofE-Cadherin or of β-Catenin is generally associated with greateraggressiveness and dedifferentiation of the tumor, in particular withrespect to gastrointestinal cancers. F. Roca et al.⁴³ have thus shownthat patients having colorectal cancer and underexpressing E-Cadherinhave a more unfavorable prognosis than patients having a normalexpression level. As early as 1983, Damsky et al.⁴⁴ showed that asoluble form of E-Cadherin could be released by the MCF-7 breast cancercell line. This soluble form corresponds to the cleavage of theextracellular portion of E-Cadherin. Later, M. Katayama et al.⁴⁵ showedthat the soluble form of E-Cadherin could be released into thebloodstream in the case of cancer, and C. Willmanns et al.⁴⁶ showed thatthe increase in the amount of E-Cadherin in the blood is correlated withthe tumor stage in colorectal cancers. A commercially available kit is,moreover, proposed by the company Takara BioChemicals (Tokyo, Japan).

The assaying of CEA (carcinoembryonic antigen) for the diagnosis ofcolorectal cancer has been proposed since 1965 by P. Gold and S.Freedman⁴⁷, but an assay for this marker in the blood has poorsensitivity for the diagnosis of colorectal cancers at a relativelynonadvanced stage. The assaying of serum CEA is thus especiallyrecommended for evaluating the risk of liver metastases⁴⁸ and fortherapeutic follow-up. In addition, it is a marker that is not veryspecific for colorectal cancer; it may in fact be increased in manyother cancers (lung, breast, etc.). On the other hand, the assaying offecal CEA appears to be more sensitive and more specific than theassaying of serum CEA or than the assaying of fecal blood⁴⁹. However,this assaying is not yet proposed routinely.

The reactive antigenic determinants 1116-NS-19-9, more commonly calledCA19-9 (carbohydrate antigen 19.9), are carried by high-molecular-weightproteins⁵⁰. The assaying of CA 19-9 in the blood is more specific thanthat of CEA. The CA 19-9 level in the blood increases in the event ofcolorectal cancer, of pancreatic cancer and of liver cancer(cholangiocarcinoma), but also in the event of noncancerous pathologicalconditions (cholangitis, etc.). Its use in combination with CEA isrecommended both at the time of diagnosis of a cancer and for follow-upof the pathological condition.

J. Holmgren et al.⁵¹ have shown that the amount of CA 50 antigen in theserum is increased in the case of colorectal cancer. The CA 50 antigenis defined by its ability to be recognized by a specific monoclonalantibody.

As regards the CA 72 marker, T. L. Klug et al.⁵² have shown that theamount of CA 72 antigen in the serum is increased in the case ofcolorectal cancer. The CA 72 antigen is defined by its ability to berecognized by a specific monoclonal antibody.

Similarly, P. Kuusela et al.⁵³ have shown that the amount of CA 242antigen in the serum is increased in the case of colorectal cancer. TheCA 242 antigen is defined by its ability to be recognized by a specificmonoclonal antibody.

The assaying of Testosterone for the diagnosis of colorectal cancer hasbeen proposed in men by M. Holland et al.⁵⁴. These authors have shown afall in the blood Testosterone level in the case of colorectal cancer.

As regards the TIMP-1, or tissue inhibitor of matrix metalloproteinasetype-1, marker, patent application US 2007/0020707 describes inparticular the assaying of TIMP-1 for the diagnosis of colorectal cancerby assaying in a body fluid.

F. Model et al.⁵⁵ showed, in July 2006, during the World Congress onGastrointestinal Cancer, that it was possible to detect methylated formsof the septin-9 gene in the plasma of patients having colorectal cancer.

M. P. Ebert et al.⁵⁶ have shown that the ALX4 gene, or aristaless-likehomeobox-4 gene, is more often methylated in the sera of patients havingcolorectal cancer than in control sera (P<0.0001). Using a thresholdvalue of 41.4 pg/mL, they have obtained a sensitivity of 83.3% and aspecificity of 70%.

Villin is described as a blood marker for the diagnosis of colorectalcancer in patent application FR2581456.

C. Bianco et al.⁵⁷ have shown that the amount of Cripto-1 in the serumis increased in the event of colorectal cancer.

The assaying of Intelectin-1 (Swiss Prot No. Q8WWA0, also known asIntestinal lactoferrin receptor, Galactofuranose-binding lectin,Endothelial lectin HL-1 or Omentin) for the diagnosis of colorectalcancer has been described in patent application US 2003/0082533.

The use of Protein Disulfide Isomerase (Swiss Prot No. P07237, alsoknown as EC 5.3.4.1, PDI, Prolyl 4-hydroxylase subunit beta, Cellularthyroid hormone-binding protein or p55), of Translationally-ControlledTumor Protein (Swiss Prot No. P13693, also known as TCTP, p23,Histamine-releasing factor, HRF or Fortilin) and of Prodefensin-A5(Swiss Prot No. Q01523), as markers in colorectal cancer, is describedrespectively in patent applications EP1724586, US 2003/0172388 and US2006/0179496. The term “(Pro)defensin” is intended to mean theprecursor, namely the Prodefensin before cleavage, the propeptide,namely the N-terminal moiety after cleavage of the Prodefensin, and themature protein, namely Defensin, corresponding to the C-terminal moietyafter cleavage.

Finally, the assaying of fecal blood is known practice and can beimplemented as previously described.

The concentration of the tumor marker chosen from group B will,depending on the marker under consideration, be increased or decreasedin the biological sample in which the method of the invention is carriedout, compared with the reference values determined for healthy patients.

Preferably, the tumor marker(s) of group B is (are) chosen from: themarkers: Beta2-Microglobulin, Proteasome 20S, Galectin-3, L-LactateDehydrogenase Chain B, Calreticulin, Regenerating Islet-Derived Protein3 Alpha, Tumor-Associated Calcium Signal Transducer 1,Epithelial-Cadherin, CEA, CA19-9, Testosterone, TIMP-1, Intelectin-1,Protein Disulfide Isomerase, Cytokeratin 20, Translationally-ControlledTumor Protein, (Pro)defensin-A5, and detection of human fecalhemoglobin.

Of course, the method of the invention may also include the detection ofany other marker for colorectal cancer that is known to those skilled inthe art.

In contrast to Ezrin which is always detected in protein form, the tumormarker(s) of interest other than Ezrin is (are) detected in the form ofa protein, or in the form of a messenger RNA, or by alteration of thecorresponding DNA (mutation or modification of methylations).

The determination of the presence, in the biological sample, of the“protein” tumor marker of interest can be carried out by any method fordetermining the presence of a protein in a sample, known to thoseskilled in the art, such as, for example, a biochemical test, includingan immunoassay, or by mass spectrometry.

The biochemical test may be any test widely known to those skilled inthe art involving molecular interactions, i.e. reactions between saidtumor marker and one or more binding partner(s) specific or not specificfor said tumor marker.

Preferably, the biochemical test is an immunoassay known to thoseskilled in the art, involving immunological reactions between the tumormarker, which is the antigen, and one or more specific bindingpartner(s), namely the antibodies directed against this antigen.

The binding partners specific or not specific for the tumor marker(s)sought in the method of the invention are any partner capable of bindingto this or these marker(s). They are said to be specific when they arecapable of binding to these markers with a high specificity, or even aspecificity of 100%. They are said to be nonspecific when theirspecificity of binding to these markers is low and they are then capableof binding to other ligands, such as proteins. By way of example,mention may be made of antibodies, antibody fractions, receptors and anyother molecule capable of binding to this marker.

The binding-partner antibodies are, for example, either polyclonalantibodies or monoclonal antibodies.

The polyclonal antibodies may be obtained by immunization of an animalwith the tumor marker concerned, followed by recovery of the desiredantibodies in purified form, by taking the serum from said animal, andseparation of said antibodies from the other serum constituents, inparticular by affinity chromatography on a column to which an antigenspecifically recognized by the antibodies, in particular said marker, isattached.

The monoclonal antibodies can be obtained by the hybridoma technique,the general principle of which is recalled hereinafter.

Firstly, an animal, generally a mouse, is immunized with the tumormarker of interest, the B lymphocytes of said animal then being capableof producing antibodies against said antigen. These antibody-producinglymphocytes are subsequently fused with “immortal” myeloma cells (murinein the example) so as to produce hybridomas. Using the heterogeneousmixture of cells thus obtained, a selection of cells capable ofproducing a particular antibody and of multiplying indefinitely is thencarried out. Each hybridoma is multiplied in the form of a clone, eachresulting in the production of a monoclonal antibody of which theproperties of recognition with respect to said tumor marker may betested, for example, by ELISA, by one-dimensional or two-dimensionalWestern blot, by immunofluorescence, or by means of a biosensor. Themonoclonal antibodies thus selected are subsequently purified, inparticular according to the affinity chromatography technique describedabove.

The monoclonal antibodies may also be recombinant antibodies obtained bygenetic engineering, by means of techniques well known to those skilledin the art.

Examples of anti-Leukocyte Elastase Inhibitor antibodies are known andare available in particular in the Abcam catalog, rabbit anti-LEIpolyclonal antibody, Cat. No. Ab47731. An anti-LEI monoclonal antibody,clone ELA-1, has been described in the article by R. Yasumatsu et al.⁵⁸.

Examples of anti-Aminoacylase 1 antibodies are known and are availablein particular in the Abnova catalog, anti-Aminoacylase 1 monoclonalantibody, clone 4F1-B7, Cat. No. H00000095-M01, and in the Abcamcatalog, chicken anti-Aminoacylase 1 polyclonal antibody, Cat. No.Ab26173.

Examples of anti-Liver Fatty Acid-Binding Protein antibodies are knownand are available in particular in the Abcam catalog, anti-L-FABPmonoclonal antibody, clone 6B6, Cat. No. Ab10059, and rabbit anti-L-FABPpolyclonal antibody, Cat. No. Ab7807.

Examples of anti-Intestinal Fatty Acid-Binding Protein antibodies areknown and are available in particular in the R&D Systems catalog,anti-I-FABP monoclonal antibody, clone 323701, Cat. No. MAB3078, and inthe Abcam catalog, rabbit anti-1-FABP polyclonal antibody, Cat. No.Ab7805.

Examples of anti-Apolipoprotein AI antibodies are known and areavailable in particular in the Biodesign Meridian Life Sciences catalog,anti-Apo AI monoclonal antibody, clone 4A90, Cat. No. H45402M and goatanti-Apo AI polyclonal antibody, Cat. No. K45252P.

Examples of anti-Apolipoprotein AII antibodies are known and areavailable in particular in the US Biological catalog, anti-Apo AIImonoclonal antibody, clone 1402, Cat. No. A2299-31C and in the BiodesignMeridian Life Sciences catalog, goat anti-Apo AII polyclonal antibody,Cat. No. K74001P.

Examples of anti-I-Plastin polyclonal antibodies are known and areavailable in particular in the Santa Cruz Biotechnology catalog. Therabbit polyclonal antibody H-300 (Cat. No. sc-28531) reacts withI-Plastin, L-Plastin and T-Plastin. The applicant has developedmonoclonal antibodies directed against I-Plastin.

Examples of anti-Beta2-Microglobulin, anti-CEA, anti-CA19-9 andanti-Testosterone antibodies are known and are in particular used in theapplicant's assay kits, respectively Vidas® β2-Microglobulin, Vidas®CEA, Vidas® CA19-9™ and Vidas® Testosterone.

Examples of anti-Proteasome 20S antibodies are known and are availablein particular in the Affinity Research Products catalog.

Examples of anti-Galectin-3, anti-L-Lactate Dehydrogenase Chain B,anti-Calreticulin, anti-Tumor-Associated Calcium Signal Transducer 1,anti-Keratin type II Cytoskeletal 8, anti-Keratin type I Cytoskeletal18, anti-Keratin type I Cytoskeletal 19, anti-Epithelial-Cadherin,anti-Villin and anti-TIMP-1 antibodies are known and are available inparticular in the Abcam catalog.

Examples of anti-Regenerating Islet-Derived Protein 3 Alpha antibodiesare known and are in particular used in the Dynabio assay kits (LaGaude, France).

Examples of anti-CA 242, anti-CA 50 and anti-CA 72-4 antibodies areknown and are available in particular in the Fujirebio catalog.

Examples of anti-Intelectin-1 antibody are known and are available inparticular in the Alexis Biochemicals catalog, anti-Intelectin-1monoclonal antibody, clone Saly-1, Cat. No. ALX-804-850-C100 and rabbitanti-Intelectin-1 polyclonal antibody, Cat. No. ALX-210-941.

Examples of anti-Protein Disulfide Isomerase antibodies are known andare available in particular in the Abcam catalog, anti-PDI monoclonalantibody, clone RL77, Cat. No. Ab5484 and rabbit anti-PDI polyclonalantibody, Cat. No. Ab3672.

Examples of anti-Cytokeratin 20 antibodies are known and are availablein particular in the Abcam catalog, anti-Cytokeratin 20 monoclonalantibody, clone Ks20.8, Cat. No. Ab962 and rabbit anti-Cytokeratin 20polyclonal antibody, Cat. No. Ab36756.

Examples of anti-TCTP antibodies are known and are available inparticular in the Abnova catalog, anti-TCTP monoclonal antibody, clone3C7, Cat. No. 157H00007178-M01 and anti-TCTP polyclonal antibody, Cat.No. 157H00007178-A01.

Examples of anti-Defensin-A5 antibodies are known and are available inparticular in the Santa Cruz Biotechnology catalog, anti-Defensin-A5monoclonal antibody, clone 8C8, Cat. No. sc-53997 and in the AlphaDiagnosis International Inc. catalog, rabbit anti-Defensin-A5 polyclonalantibody, Cat. No. HDEFA51-A.

The binding partners which are specific or not specific for the tumormarker(s) sought in the method of the invention may be used as a capturereagent, as a detection reagent or as capture and detection reagents.

The visualization of the immunological reactions, i.e. of the tumormarker/binding partner binding, may be carried out by any means ofdetection, such as direct or indirect means.

In the case of direct detection, i.e. without the involvement of alabel, the immunological reactions are observed, for example, by surfaceplasmon resonance or by cyclic voltammetry on an electrode bearing aconductive polymer.

The indirect detection is carried out by means of labeling, either ofthe “revealing reagent” binding partner, or of the tumor marker ofinterest itself. In the latter case, this is then described as acompetition method.

The term “labeling” is intended to mean the attachment of a labelreagent capable of directly or indirectly generating a detectablesignal. A nonlimiting list of these label reagents comprises:

-   -   enzymes which produce a signal that can be detected, for        example, by colorimetry, fluorescence or luminescence, such as        horseradish peroxydase, alkaline phosphatase, β-galactosidase or        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent or luminescent compounds or        dyes,    -   radioactive molecules such as ³²P, ³⁵S or ¹²⁵I, and fluorescent        molecules such as Alexa or phycocyanins.

Indirect detection systems may also be used, such as, for example,ligands capable of reacting with an antiligand. Ligand/antiligand pairsare well known to those skilled in the art, this being the case, forexample, of the following pairs: biotin/streptavidin, hapten/antibody,antigen/antibody, peptide/antibody, sugar/lectin,polynucleotide/sequence complementary to the polynucleotide. In thiscase, it is the ligand which carries the binding partner. The antiligandmay be directly detectable by means of the label reagents described inthe previous paragraph, or may itself be detectable by means of aligand/antiligand.

These indirect detection systems may result, under certain conditions,in an amplification of the signal. This signal amplification techniqueis well known to those skilled in the art, and reference may be made tothe prior patent applications FR98/10084 or WO-A-95/08000 by theapplicant or to the article by Chevalier et al.⁵⁹.

Depending on the type of labeling used, those skilled in the art willadd reagents that make it possible to visualize the labeling.

By way of example of immunoassays as defined above, mention may be madeof “sandwich” methods such as ELISA, IRMA and RIA, “competition” methodsand direct immunodetection methods such as immunohistochemistry,immunocytochemistry, Western blotting and Dot blotting.

Mass spectrometry can also be used for detecting, in the biologicalfluid, the tumor marker(s) sought in the method of the invention. Theprinciple of spectrometry is widely known to those skilled in the artand is described, for example, in Patterson, S.⁶⁰.

To do this, the biological sample, which may or may not have beenpretreated, is passed through a mass spectrometer and the spectrumobtained is compared with that of the tumor marker(s) sought in themethod of the invention. An example of pretreatment of the sampleconsists in passing it over an immunocapture support comprising one ofthe binding partners for the tumor marker(s) sought in the method of theinvention, for example an antibody directed against the tumor marker(s)sought in the method of the invention. Another example of pretreatmentof the sample may be prefractionation of the biological sample in orderto separate the proteins of the sample from one another. In techniqueswell known to those skilled in the art, the predominant proteins of thesample may, for example, first of all be depleted.

When the method of the invention is a “sandwich” method, it may use twomonoclonal antibodies or alternatively one monoclonal antibody and onepolyclonal antibody. Of course, in the latter case, the monoclonalantibody is at least an anti-Ezrin monoclonal antibody directed againstan Ezrin epitope chosen from the epitopes of sequence SEQ ID No. 1, SEQID No. 2, SEQ ID No. 3, SEQ ID No. 4+SEQ ID No. 5, SEQ ID No. 6+SEQ IDNo. 7 and SEQ ID No. 8.

Examples of anti-Ezrin antibodies are known and are available inparticular in the Abcam catalogue, for example rabbit anti-Ezrinpolyclonal antibody, Cat. No. Ab47418.

According to one embodiment of the invention, the method of theinvention uses two monoclonal antibodies, preferably at least oneanti-Ezrin monoclonal antibody directed against an epitope of sequenceSEQ ID No. 1 and at least one other monoclonal antibody directed againstan Ezrin epitope chosen from the epitopes of sequences SEQ ID No. 2, SEQID No. 4+SEQ ID No. 5 and SEQ ID No. 6+SEQ ID No. 7. More preferably,the method of the invention uses at least one anti-Ezrin antibodydirected against an epitope of sequence SEQ ID No. 1 and at least oneother antibody directed against an epitope of sequence SEQ ID No. 4+SEQID No. 5.

Moreover, when the method of the invention is immunohistochemistry, itis preferred to use at least one anti-Ezrin monoclonal antibody directedagainst an epitope of sequence SEQ ID No. 2, this constituting aparticular embodiment of the invention.

The determination of the presence, in the biological sample, of the“mRNA” tumor marker of interest may be carried out by any method fordetermining the presence of mRNA in a sample, namely either directdetection of the mRNA, or indirect detection of the mRNA, or any othermethod for determining the presence of an RNA in a sample, known tothose skilled in the art.

The term “direct detection of the mRNA” is intended to mean thedemonstration of the mRNA itself in the biological sample.

The direct detection of the mRNA in the biological sample may be carriedout by any means known to those skilled in the art, such as, forexample, by hybridization with a binding partner specific for the mRNA,where appropriate after amplification by the PCR or NASBA technique.

The term “hybridization” is intended to mean the process during which,under suitable conditions, two nucleotide fragments bind to one anotherwith stable and specific hydrogen bonds so as to form a double-strandedcomplex. These hydrogen bonds form between the complementary basesadenine (A) and thymine (T) (or uracil (U)) (referred to as an A-T bond)or between the complementary bases guanine (G) and cytosine (C)(referred to as a G-C bond). The hybridization of two nucleotidefragments may be complete (reference is then made to complementarynucleotide fragments or sequences), i.e. the double-stranded complexobtained during this hybridization comprises only A-T bonds and C-Gbonds. This hybridization may be partial (reference is then made tosufficiently complementary nucleotide fragments or sequences), i.e. thedouble-stranded complex obtained comprises A-T bonds and C-G bonds whichmake it possible to form the double-stranded complex, but also basesthat are not bonded to a complementary base. The hybridization betweentwo nucleotide fragments depends on the operating conditions that areused, and in particular the stringency. The stringency is defined inparticular as a function of the base composition of the two nucleotidefragments, and also by the degree of mismatching between two nucleotidefragments. The stringency can also depend on the reaction parameters,such as the concentration and the type of ionic species present in thehybridization solution, the nature and the concentration of denaturingagents and/or the hybridization temperature. All these data are wellknown and the appropriate conditions can be determined by those skilledin the art. In general, depending on the length of the nucleotidefragments that it is desired to hybridize, the hybridization temperatureis between approximately 20 and 70° C., in particular between 35 and 65°C., in a saline solution at a concentration of approximately 0.5 to 1 M.The binding partners which are specific or not specific for the mRNA areany partner capable of binding to this mRNA. By way of example, mentionmay be made of nucleic probes, amplification primers, and any othermolecule capable of binding to this mRNA.

The term “hybridization probe” is intended to mean a nucleotide fragmentcomprising from 5 to 100 nucleic units, in particular from 10 to 35nucleic units, having a hybridization specificity under given conditionsso as to form a hybridization complex with the material specific for thetarget gene of interest. The hybridization probe may comprise a labelenabling its detection.

For the purpose of the present invention, the term “amplificationprimer” is intended to mean a nucleotide fragment comprising from 5 to100 nucleic units, preferably from 15 to 30 nucleic units, enabling theinitiation of an enzymatic polymerization, such as, in particular, anenzymatic amplification reaction. The term “enzymatic amplificationreaction” is intended to mean a process that generates multiple copiesof a nucleotide fragment via the action of at least one enzyme. Suchamplification reactions are well known to those skilled in the art andmention may in particular be made of the following techniques:

PCR (polymerase chain reaction), as described in U.S. Pat. No.4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159,

NASBA (nucleic acid sequence-based amplification) with patentapplication WO 91/02818, and

TMA (transcription mediated amplification) with U.S. Pat. No. 5,399,491.

The term “detection” is intended to mean either a physical method, or achemical method with an intercalating dye such as SYBR® Green I orethidium bromide, or a method of detection using a label. Many detectionmethods exist for detecting nucleic acids⁶². The appropriate labels areas defined above.

For the purpose of the present invention, the hybridization probe may bea “detection” probe. In this case, the “detection” probe is labeled bymeans of a label as defined above. By virtue of the presence of thislabel, it is possible to detect the presence of a hybridization reactionbetween a given detection probe and the transcript to be detected.

The detection probe may in particular be a “molecular beacon” detectionprobe⁶³. These “molecular beacons” become fluorescent duringhybridization. They have a stem-loop structure and contain a fluorophoreand a quencher group. The binding of the specific loop sequence with itscomplementary target nucleic acid sequence causes unfolding of the stemand the emission of a fluorescent signal during excitation at theappropriate wavelength.

The hybridization probe may also be a “capture” probe. In this case, the“capture” probe is immobilized or can be immobilized on a solid supportby any appropriate means, i.e. directly or indirectly, for example bycovalence or adsorption. The appropriate solid supports are known tothose skilled in the art, and, by way of examples, mention may be madeof synthetic materials or natural materials, latices, magneticparticles, metal derivatives, gels, etc. The solid support may be in theform of a microtitration plate, a membrane as described in applicationWO-A-94/12670 or a particle. It is also possible to immobilize severaldifferent capture probes on the support, each capture probe beingspecific for a target transcript. In particular, it is possible to use,as support, a biochip on which a large number of probes may beimmobilized.

The immobilization of the probes on the support is also known to thoseskilled in the art, and mention may be made of a deposit of probes bydirect transfer, microdeposition, in situ synthesis andphotolithography.

The demonstration, in the biological sample, of the DNA modifications oranomalies in the gene encoding the tumor marker of interest may becarried out by any method for determining DNA alterations in a sample,namely either the direct detection of mutations, or the demonstration ofalterations in the methylation profile of the loci of interest, or anyother method for determining DNA alterations in a sample, known to thoseskilled in the art.

The mutations may include point substitutions of one nucleotide withanother, deletions of one or more nucleotides and insertions of one ormore nucleotides. The mutations may be located in the coding portion ofthe gene of the tumor marker of interest, or in the 5′ and 3′ noncodingportions, such as the transcription promoter region or the transcriptiontermination region.

The strategies for demonstrating a mutation are based on molecularbiology techniques and comprise steps of DNA extraction, amplificationby PCR or another amplification technique, hybridization and/orsequencing. In the case of colorectal cancer, the following method hasbeen successfully used to detect mutations in fecal DNA: concentrationof the DNA by precipitation, enrichment in the target using captureoligonucleotides on magnetic beads, PCR amplification of the genes ofinterest, solid-phase sequencing for identifying point mutations⁶⁴. Thedeletions were identified with respect to the difference in size betweenthe expected reference fragment and the mutated fragment. Imperiale etal.⁶⁴ have described a panel of 21 mutations located in the K-ras, APCand p53 genes, which makes it possible to detect 16/31 of invasivecancers.

Other DNA markers used are the BAT-26 deletion, which is a marker forinstability of microsatellites and highly amplifiable DNA called longDNA (L-DNA), which is not a specific marker but which appears to reflectthe disorganized apoptosis of exfoliated tumor cells in the coloniclumen⁶⁵. These markers are not satisfactory, either in terms of theirsensitivity or in terms of their specificity.

As previously indicated, the DNA alterations may also correspond to amodification of the methylation profile of the gene corresponding to thetumor marker of interest. The modification of the methylation profilemay correspond to a hypomethylation (decrease in the number ofmethylations) or to a hypermethylation (increase in the number ofmethylations). The altered units may be located in the coding portion ofthe gene of the tumor marker of interest, or in the 5′ and 3′ noncodingportions, such as the transcription promoter region or the transcriptiontermination region.

The analysis of the DNA methylation may be carried out using techniquesbased on qualitative and/or quantitative PCR, such as MSP(methylation-specific PCR), bisulfite sequencing, digestion with amethylation-sensitive restriction enzyme coupled with PCR, COBRA(combined bisulfite restriction analysis) and Ms-SNuPE(methylation-sensitive single nucleotide primer extension). All thesetechniques have been reviewed comparatively and in detail in amethodology article⁶⁶.

In the literature, several hypermethylated genes have been reported inthe case of colorectal cancer. By way of example, mention may be made ofthe ALX4 (aristaless-like homeobox-4) gene⁵⁶, the promoter region of theTPEF/HHP1 (transmembrane protein containing epidermal growth factor andfollistatin domain) gene⁶⁷ or else the septin-9 gene⁶⁸.

When, in the method of the invention, at least two markers are detected,they may be demonstrated separately, for example using differentimmunoassay measurements, or else simultaneously, in a multiplex assay.

When, in the method of the invention, two markers of different natureare detected, for example a protein marker and an mRNA marker, twodifferent detection methods, chosen from those described above, may beused. They may also be detected simultaneously, in the same detectionmedium and under the same reaction conditions, as described in patentapplication WO 03/104490. The steps of the detection method described inthis patent application, which consists in simultaneously detectinghybridization and immunological reactions in a sample that may containtarget analytes constituted of at least one nucleic acid and of at leastone other ligand of different nature, consist in:

(i) depositing a known volume amount of the sample diluted in a reactionbuffer, on a capture surface precoated with capture partners for saidtarget analytes, said capture partners comprising at least one nucleicprobe and at least one antiligand,

(ii) reacting at a temperature of between 15° C. and 60° C., and

(iii) visualizing the hybridization and immunological reactions thusobtained.

The biological sample may require a particular treatment because it maycontain the tumor marker(s) sought in the method of the invention, assuch, or else it may contain circulating tumor cells which contain themarkers sought in the method of the invention and/or circulating tumorcells which are capable of secreting the marker(s) sought in the methodof the invention.

Thus, according to one embodiment of the invention, the biologicalsample is pretreated in order to isolate the circulating tumor cellscontained in said fluid.

The expression “isolate circulating tumor cells” is intended to meanobtain a cell fraction enriched in circulating tumor cells.

The treatment of the biological sample in order to isolate thecirculating tumor cells can be carried out by cell sorting in a flowcytometer, by enrichment on Ficoll, by enrichment with magnetic beadscovered with specific antibodies, or by any other method of specificenrichment known to those skilled in the art.

In the case of blood as biological sample, the circulating tumor cellsmay be isolated by means of a technique of cell separation on Ficollcombined with depletion of the blood cells using anti-CD45 antibodiescoupled to magnetic beads (Dynal Biotech ASA, Norway).

The detection of the tumor marker(s) sought in the method of theinvention can then be carried out directly using circulating tumor cellsisolated from the biological sample, for example by immunocytochemicallabeling of these cells with an antibody against tumor marker(s) soughtin the method of the invention, after having deposited the circulatingtumor cells on a slide by cytospin. The detection of the tumor marker(s)sought in the method of the invention may also be carried out directlyin the circulating tumor cells using the flow cytometry method asdescribed in Métézeau et al.⁶⁹.

Under these conditions, said circulating cells can be treated underconditions which make it possible to block the tumor marker(s) sought inthe method of the invention, inside said cells. Such a treatment isdescribed by Mathieu et al.⁷⁰.

The detection of the tumor marker(s) sought in the method of theinvention is then carried out after having made the cell membranepermeable so as to allow entry of the binding partners specific for themarker(s) sought in the method of the invention.

The direct detection of the tumor marker(s) used in the method of theinvention, based on the circulating cells, may also be carried out bymeans of an ELISPOT method, for example by means of the method describedin patent application WO 03/076942 filed by the applicant. This methodis a method for detecting and/or quantifying circulating tumor cells ofa biological sample, which are capable of releasing or secreting, invitro, one or more tumor marker(s), comprising the steps consisting in:

(i) depositing an amount of said cells at the bottom of a culturesurface to which at least one binding partner specific for said tumormarker(s) is attached,

(ii) culturing said cells under conditions such that they release orsecrete said tumor markers, which are immunocaptured at the bottom ofthe culture surface,

(iii) removing the cells by washing,

(iv) adding at least one labeled conjugate specific for said tumormarkers, and

(v) visualizing the labeling thus obtained.

The direct detection of the tumor marker(s) used in the method of theinvention in the tumor cells may also be carried out in the culturemedium of said cells after having cultured them under conditions suchthat they secrete tumor marker(s) used in the method of the invention.

The culture conditions for release or the expression of the tumormarkers are conventional conditions such as 37° C. under a humidatmosphere and at 5% CO₂.

When the biological sample is a solid sample, the presence of the tumormarker(s) may also be shown in vivo, in situ in the tumors.

In order to show the presence of a tumor marker in a tumor in vivo, anyimaging method known to those skilled in the art may be used. For this,a binding partner for said tumor marker may be coupled to an imagingtracer.

The term “coupling of the binding partners to an imaging tracer” isintended to mean the attachment of a tracer capable of being detected byany imaging method known to those skilled in the art, and of directly orindirectly generating a detectable signal. Thus, the tracer may be aradioactive tracer such as technetium-99. In this case, the organ whichhas the primary cancer or the metastases will bind the tumor marker andits tracer. The radiation emitted by the organ can be filmed with aspecial camera, for example a gamma-camera. The instrument collects thescintillations generated by the radioactive substance and thus makes itpossible to visualize the organ.

In another method of the invention, the tracer may comprise apositron-emitting radioactive substance (fluorine 18). The images willthen be acquired by a positron emission tomography system.

In another preferred method of the invention, the partner of the tumormarker(s) may be coupled to nanoparticles. By way of example, they maybe supramagnetic nanoparticles; for example, anionic magneticnanoparticles for use in direct cell labeling and in vivo detection bynuclear magnetic resonance imaging. They may also be gold nanoparticles.

By virtue of the methods of the invention which enable the detection ofthe tumor marker in vivo, the areas of the body where there has beenbinding of the binding partner for the tumor marker, cancers producingtumor marker, and in particular colorectal cancer, and alsolocalizations of their remote metastases and involved lymph nodes, maybe visualized.

The method of the invention may be used not only for early diagnosis butalso for screening, therapeutic follow-up, prognosis and relapsediagnosis in relation to colorectal cancer, since only the cancer cellssecrete Ezrin and this production depends on the grade of the cancer,which constitutes another subject of the invention.

In addition, the epitopes described in this application, having thesequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4+SEQ IDNo. 5, SEQ ID No. 6+SEQ ID No. 7 or SEQ ID No. 8, are novel and formanother subject of the invention.

The invention will be understood more clearly by means of the followingexamples which are given by way of nonlimiting illustration, and also bymeans of the attached FIGS. 1 to 21, in which:

FIG. 1 is a graph relating to the assaying by ELISA of LEI, in ng/ml, inthe serum of patients having colorectal cancer (CRC+) and of healthypatients (CRC−),

FIG. 2 is a graph relating to the assaying by ELISA of Ezrin, in ng/ml,in the serum of patients having colorectal cancer (CRC+) and of healthypatients (CRC−),

FIG. 3 is a graph relating to the assaying by ELISA of Aminoacylase 1,in ng/ml, in the serum of patients having colorectal cancer (CRC+) andof healthy patients (CRC−),

FIG. 4 is a graph relating to the assaying by ELISA of L-FABP, in ng/ml,in the serum of patients having colorectal cancer (CRC+) and of healthypatients (CRC−),

FIG. 5 is a graph relating to the assaying by ELISA of I-FABP, in pg/ml,in the serum of patients having colorectal cancer (CRC+) and of healthypatients (CRC−),

FIG. 6 is a graph relating to the assaying by ELISA of ApolipoproteinAI, in μg/ml, in the serum of patients having colorectal cancer (CRC+)and of healthy patients (CRC−), either by microplate ELISA (FIG. 6A), orwith the Lincoplex kit (FIG. 6B),

FIG. 7 is a graph relating to the assaying, using the Linco multiplexkit, of Apolipoprotein AII, in μg/ml, in the serum of patients havingcolorectal cancer (CRC+) and of healthy patients (CRC−),

FIG. 8 is a graph relating to the assaying by ELISA of I-Plastin, in RFV(relative fluorescence value), in the serum of patients havingcolorectal cancer (CRC+) and of healthy patients (CRC−),

FIG. 9 is a graph relating to the assaying by ELISA ofBeta2-Microglobulin, in ng/ml, in the serum of patients havingcolorectal cancer (CRC+) and of healthy patients (CRC−),

FIG. 10 is a graph relating to the assaying by ELISA of CEA, in ng/ml,in the serum of patients having colorectal cancer (CRC+) and of healthypatients (CRC−),

FIG. 11 is a graph relating to the assaying by ELISA of CA 19-9, inU/ml, in the serum of patients having colorectal cancer (CRC+) and ofhealthy patients (CRC−),

FIG. 12 is a graph relating to the assaying by ELISA of Testosterone, inng/ml, in the serum of patients having colorectal cancer (CRC+) and ofhealthy patients (CRC−),

FIG. 13 is a graph relating to the assaying by ELISA of E-Cadherin, inng/ml, in the serum of patients having colorectal cancer (CRC+) and ofhealthy patients (CRC−),

FIG. 14 is a graph relating to the assaying by ELISA of PAP1, in ng/ml,in the serum of patients having colorectal cancer (CRC+) and of healthypatients (CRC−),

FIG. 15 is a graph relating to the assaying by ELISA of Galectin-3, inRFV, in the serum of patients having colorectal cancer (CRC+) and ofhealthy patients (CRC−),

FIG. 16 is a graph relating to the assaying by ELISA of LDH, in ng/ml,in the serum of patients having colorectal cancer (CRC+) and of healthypatients (CRC−),

FIG. 17 is a graph relating to the assaying by ELISA of Proteasome 20S,in ng/ml, in the serum of patients having colorectal cancer (CRC+) andof healthy patients (CRC−),

FIG. 18 is a graph relating to the assaying by ELISA of Aminoacylase 1,in ng/ml, in the stools of patients having colorectal cancer (CRC+) andof healthy patients (CRC−),

FIG. 19 is a graph relating to the assaying by ELISA of Galectin-3, inRFV, in the stools of patients having colorectal cancer (CRC+) and ofhealthy patients (CRC−),

FIG. 20 is a graph relating to the assaying by ELISA of Proteasome 20S,in RFV, in the stools of patients having colorectal cancer (CRC+) and ofhealthy patients (CRC−), and

FIG. 21 is a graphic representation of an ELISPOT assay for LEI, forEzrin and for Galectin-3, in number of spots per 10⁶ cancer cells of theCaco-2, HT-29 and HT29-B6 lines.

EXAMPLE 1 Cloning of the Genes Encoding the Tumor Markers and Expressionof the Recombinant Proteins

1. cDNA Amplification and Cloning

The Caco-2 colorectal cancer line is cultured in DMEM medium containing2 mM of L-glutamine, without FCS (fetal calf serum) (all Gibco).

For the cloning of the LEI, L-FABP and Gal-3 genes, the messenger RNAswere extracted from a pellet of 10⁸ Caco-2 cells using the InvitrogenFastTrack 2.0 kit (Cat. No. 45-0019) according to the protocol suppliedby the manufacturer. The reverse transcription and PCR steps are carriedout in a single step using 450 ng of Caco-2 mRNA with the SuperscriptIII One Step RT-PCR System kit (Invitrogen Cat. No. 12574-018) using thePlatinum Taq DNA polymerase enzyme according to the protocol supplied bythe manufacturer. The PCR primers used for the gene amplification aregiven in Table 1.

TABLE 1 Genes and primers Oligonucleotides LEI OL215 (SEQ ID No. 9)5′-ATGGAGCAGCTGAGCTCAGCAAAC-3′ OL216 (SEQ ID No. 10)5′-CTAAGGGGAAGAAAATCTCCCCAA-3′ L-FABP Forward (SEQ ID No. 11)5′-CGGAGCGTCTCCCATGAGTTTCTCCGGCAAGTA-3′ Reverse (SEQ ID No. 12)5′-GAAATGCAGACTTGTCTAGATGCGCTTGCTGATG CGCTTGAAGACAATG-3′ Gal-3 OL217(SEQ ID No. 13) 5′-ATGGCAGACAATTTTTCGCTCC-3′ OL218 (SEQ ID No. 14)5′-TTATATCATGGTATATGAAGCACTGG-3′

The DNA fragments obtained were cloned into the vector pCR2.1 TOPO (LEIand Gal-3) with the TA cloning kit (Invitrogen Cat. No. K4520-01) or thevector pCMV6-XL4 from Origene (L-FABP) after digestion with Bsm BI andXba I. The plasmids were sequenced in order to verify that the cDNAindeed complies with the expected sequence.

For the cloning of the gene encoding Aminoacylase 1, the total RNA wasextracted from a pellet of 10⁸ Caco-2 cells using the RNA Easy Mini kitfrom Qiagen, according to the protocol supplied by the manufacturer. Thereverse transcription is carried out using 10 ng of Caco-2 RNA, with theSuperscript II enzyme (Invitrogen) according to the protocol supplied bythe manufacturer. The reverse transcription primer is an oligo(dT).

The cDNA derived from this reaction was used as template in a PCRreaction using the AccuPrime Pfx kit (Invitrogen Cat. No. 12344-024)according to the protocol supplied by the manufacturer. The PCR primersare: ACY-1 Fwd2 (SEQ ID No. 15:5′-GCGAATTCTTTAAGAAGGAGATATACATATGACGAGCAAAGGTCCGGAAGAGGAGCACCCATCG-3′)and ACY-1 Rev (SEQ ID No. 16: 5′-GCAAGCTTCAGCTGTCACTGGGCAGGGC-3′).

Under these conditions, it was possible to amplify a 1.3 kb fragmentwhich was cloned into a cloning vector of the Zero Blunt TOPO PCRcloning kit type (Invitrogen Cat. No. K2820-20). This plasmid wassequenced in order to verify that the cDNA indeed complies with theexpected sequence.

The following DNA fragment (SEQ ID No. 17) containing the I-FABP openreading frame was obtained by chemical synthesis, carried out by thecompany Geneart.

SEQ ID No. 17: GGTACCGAATTCCGCGTTTGACAGCACTTGGAAGGTAGACCGGAGTGAAAACTATGACAAGTTCATGGAAAAAATGGGTGTTAATATAGTGAAAAGGAAGCTTGCAGCTCATGACAATTTGAAGCTGACAATTACACAAGAAGGAAATAAATTCACAGTCAAAGAATCAAGCGCTTTTCGAAACATTGAAGTTGTTTTTGAACTTGGTGTCACCTTTAATTACAACCTAGCAGACGGAACTGAACTCAGGGGGACCTGGAGCCTTGAGGGAAATAAACTTATTGGAAAATTCAAACGGACAGACAATGGAAACGAACTGAATACTGTCCGAGAAATTATAGGTGATGAACTAGTCCAGACTTATGTGTATGAAGGAGTAGAAGCCAAAAGGATCTTTAAAAAGGATTCTAGAGTCGACGAGCTC.

2. Expression Vector Construction

The genes encoding LEI and Galectin-3 were subcloned into theprokaryotic expression vector pMR78⁷¹ and the L-FABP gene was clonedinto the vector pET3d (New England Biolabs). The restriction sitesnecessary for the cloning were introduced by PCR using, as template, theplasmids pCR2.1 TOPO-LEI, pCR2.1 TOPO-Gal-3 and pCMV6-LFABP. The PCRenzyme is the Promega Pfu DNA polymerase, the PCR reaction was carriedout according to the manufacturer's instructions, with the primers givenin Table 2.

TABLE 2 Genes and primers Oligonucleotides LEI OL228 (SEQ ID No. 18)5′-ATGGGAATTCAGGAGCAGCTGAGCTCAGCAA-3′ OL229 (SEQ ID No. 19)5′-CGATAAGCTTAAGGGGAAGAAAATCTCCCC-3′ L-FABP Forward (SEQ ID No. 20)5′-GCTGGCCATGGGCAGCAGCCATCATCATCATCATCA CATGAGTTTCTCCGGCAAGTACCAAC-3′Reverse (SEQ ID No. 21) 5′-GCACGGATCCTAGATGCGCTTGCTGATGCGCTTGAA GAC-3′Gal-3 OL230 (SEQ ID No. 22) 5′-ATGGGAATTCAGGCAGACAATTTTTCGCTCC-3′ OL231(SEQ ID No. 23) 5′-CGATAAGCTTATATCATGGTATATGAAGCACTGG-3′

The PCR products containing the open reading frames encoding LEI orGalectin-3 were digested with the Eco RI and Hind III restrictionenzymes. The fragments were introduced into the vector pMR78 restrictedwith the same enzymes (plasmids pMR-LEI and pMR-Gal-3). The vector pMR78contains a 6-histidine sequence in frame with the protein to beexpressed, which enables purification by metal-chelate affinitychromatography. The L-FABP PCR product was cloned into the vector pET3d,at the Nco I and Bam HI restriction sites.

For Aminoacylase 1, the TOPO cloning vector was directly digested withthe Eco RI and Hind III restriction enzymes in order to generate a 1.3kb fragment containing the acyl open reading frame, which was introducedinto the vector pStaby1 (Eurogentec). The recombinant plasmid is calledpStaby1-ACY.

For I-FABP, the cloning vector provided by Geneart was digested with theEco RI and Sal I restriction enzymes in order to generate anapproximately 400 by fragment containing the coding sequence, which wasintroduced into the vector pMRCH79 (derived from the vector pMR78,bioMérieux). The recombinant plasmid is called pMRCH-IFABP.

The plasmids pGEX-Ezrine and pGEX-1-plastin, which make it possible toexpress, respectively, Ezrin and I-Plastin fused with GST (glutathioneS-transferase), were supplied by the Institut Curie.

3. Recombinant Protein Expression and Purification

The expression plasmids for producing the recombinant tumor markers areintroduced into E. coli BL21 bacteria and derivatives (Stratagene). Thecultures are carried out at ambient temperature with shaking. Theprecise culture conditions for each protein are reproduced in Table 3.IPTG is isopropyl-beta-D-1-thiogalactosidase.

The bacterial pellets are taken up in 2×PBS (phosphate buffered saline)buffer and passed through a cell disintegrator at 1.5 kbar (ConstantSystem). The lysates are centrifuged at 3000 g for 30 min at 4° C. Thesupernatant contains the soluble proteins. The pellet contains theinclusion bodies. The buffer for solubilizing the inclusion bodiesdepends on the protein.

For LEI, the purification is carried out using the soluble fraction, ona column containing 5 mL of Ni-NTA-Sepharose resin (Qiagen) and theprotein is eluted with 2×PBS containing 450 mM imidazole, pH 7.5.

For Galectin-3, the inclusion bodies are solubilized in 2×PBS containing1M urea, and passed over 5 mL of Ni-NTA-Sepharose resin (Qiagen) and theGal-3 protein is eluted with 2×PBS containing 450 mM imidazole and 1Murea, pH 7.5.

For L-FABP, the purification is carried out using the soluble fraction,with the Ni-IDA kit from Macherey-Nagel.

TABLE 3 Culture IPTG Strain volume induction Purification LEI BL21 250mL 0.1 mM Ni-NTA Gal-3 BL21-Codon 400 mL 0.5 mM Ni-NTA plus (DE3)-RIPLL-FABP BL21 500 mL 0.1 mM Ni-IDA GST-Ezrin BL21 250 mL 0.1 mM GST ACY-1BL21-Codon 500 mL 0.1 mM other plus (DE3)-RIPL

For GST-Ezrin, the purification is carried out using the inclusionbodies solubilized in 100 mM Tris buffer containing 8M urea and 10 mMDTT, by GST affinity chromatography. A column containing 5 mL ofGlutathione Sepharose 4 fast flow gel (Amersham) is used. Theequilibration and washing buffer is 2×PBS containing 0.05% Tween 20. Theelution buffer is 50 mM Tris-HCl containing 20 mM reduced glutathione,pH 8.

For Aminoacylase 1, the soluble fraction of the culture is passed overan Amersham HiTrap Q FF column and the ACY-1 protein was eluted with0.3M NaCl at pH 7.5. Since several other proteins were co-eluted underthese conditions, the purification was continued on ahydrophobic-interaction column (HIC Phenyl HP, Amersham). The ACY-1protein was eluted with 0.5M NaCl at pH 7.

The recombinant GST-1-Plastin protein was provided by the Institut Curiein purified form.

The recombinant Calreticulin protein was produced by the company ProteusServices for Industry (Dijon, France). The sequence encodingCalreticulin was obtained by chemical synthesis.

EXAMPLE 2 Production of Monoclonal Antibodies Directed Against the TumorMarkers

1. Animal Model

The immunization experiments were carried out in female BALB/c (H-2^(d))mice that were 6 to 8 weeks at the time of the first immunization.

2. Immunogens and Immunizations

In order to increase the immune responses obtained in the mice and to beable to generate monoclonal antibodies, the tumor markers were producedin the form of recombinant proteins produced according to the proceduresdescribed in Example 1. The LDH protein was obtained from the companySciPac (Cat. No. 103-133). These proteins were mixed volume for volumewith Freund's adjuvant (Sigma), prepared in the form of a water-in-oilemulsion and which is known to have a good immunogenic capacity. 3 micewere immunized for each tumor marker. The mice received 3 successivedoses of 10 μg of the immunogens at 0, 2 and 4 weeks. All the injectionswere given subcutaneously. The first injection is given as a mixturewith complete Freund's adjuvant, the following two are given as amixture with incomplete Freund's adjuvant. Between D50 and D70 after thefirst injection, the humoral responses were restimulated with anintravenous injection of 100 μg of the recombinant protein.

3. Monitoring of the Appearance of the Humoral Response

In order to monitor the appearance of the antibodies, blood samples aretaken regularly from the mice. The presence of the anti-tumor markerantibodies is tested using an ELISA. The protein of interest is used forcapture (1 μg/well); after saturation, the antigen is reacted withvarious dilutions of the test sera (incubation at 37° C. for 1 h). Thespecific antibodies present in the serum are revealed with an AffiniPuregoat anti-mouse IgG antibody conjugated to alkaline phosphatase (H+L,Jackson Immunoresearch, Cat no. 115-055-146), which binds to theantibodies being sought (0.1 μg/well).

4. Production of Monoclonal Antibodies

Three days after the final injection, for each tumor marker, one of theimmunized mice was sacrificed. The blood and the spleen were taken. Thesplenocytes obtained from the spleen were cultured with Sp2/0-Ag14myeloma cells in order for them to fuse and become immortalized,according to the protocol described by Köhler and Milstein^(72,73).After an incubation period of 12-14 days, the supernatants of thehybridomas obtained were screened in order to determine the presence ofanti-tumor marker antibodies, using the ELISA assay described in point 3of this example. When GST fusion proteins were used as immunogen, theclones directed against GST are eliminated by carrying out an ELISAscreening with uncoupled GST for capture. The positive hybridomacolonies were subcloned twice according to the limiting dilutiontechnique, which is well known to those skilled in the art.

5. Characterization of the Monoclonal Antibodies by Immunoblotting

The list of monoclonal antibodies obtained against the various tumormarkers is given in Table 4. These monoclonal antibodies were analyzedby the Western blotting technique.

TABLE 4 Tumor markers Monoclonal antibody name Leukocyte ElastaseInhibitor (LEI) 21B10A5 and 10E1H1 Ezrin 4A7A6C1, 4A9H5, 4D9B9, 4G11B5,5G2D12, 8A8G6, 8B5G6G4, 9D2A11 Aminoacylase 1 2A7F6 and 11H7D9 I-plastin3D11D10, 8C8C5, 3A3H2, 8G2D2 Calreticulin 5C10H10 and 11B6D11 L-lactatedehydrogenase chain B 3F11E11 and 12F10G8 (LDH) Galectin-3 12F8A12 and14A5G1

5.1. Methodology

The Caco-2 and HT-29 line cell culture extracts are prepared by directlylyzing the cell pellet with 600 μl of an aqueous solution of 8.3M urea,2M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 mM DTT, 2% Servalyte 4-9 (Serva, Heidelberg,Germany) and 0.1 g/l Orange G, and then treated according to the NuPAGENovex gel sample preparation protocol (Invitrogen). To obtain the tissueextracts, tumor and mucosal biopsies of patients GHBD001, GHBD004 andCLSP109 were dissected with a scalpel, and were then subjected to 10cycles of extraction in the Medimachine system (Becton Dickinson) using50-μm Medicons with 1 ml of PBS buffer containing 2.5 mM EDTA andprotease inhibitors (tablets, Roche). These 10 ml of cell suspension arepooled, made up to 25 ml, and then centrifuged for 15 min at 600 g. Thesupernatant corresponds to the tissue extract which is treated accordingto the NuPAGE Novex gel sample preparation protocol. Reduced samples areused, at a final total protein concentration of 0.4 mg/ml. The depositvolume is 20 μl per well, on a NuPAGE Novex Bis-Tris 4-12% gel, withMOPS running buffer. After migration (at 200 V, for 1 hour) and transferonto a PVDF membrane (at 400 mA, for 45 min), the quality of thetransfer is assessed by staining with amido black.

The membranes are saturated with 5% skimmed milk (Régilait) in asolution of TNT (15 mM Tris, 0.14M NaCl, 0.5% Tween 20, pH 8) at ambienttemperature for 1 hour. After saturation, the membranes are incubatedfor 1 hour with the various test antibodies diluted to 10 μg/ml in thesaturating solution. After rinsing with TNT, the membranes are incubatedfor 1 hour at ambient temperature with an anti-mouse-horseradishperoxidase conjugate diluted to 1:5000, (Cat No. 115-035-062, JacksonImmunoresearch) in the saturating solution. After rinsing, thedeveloping is carried out with the Substrate Supersignal West DuraExtended kit (Cat No. 34076, Pierce) according to the recommendedinformation for use.

The chemiluminescence signal on the membranes was measured with theVersaDoc imaging system from Biorad. Based on the image of the Westernblot, the volumes of the bands which correspond to the various tumormarkers were evaluated with the QuantityOne software (Bio-Rad). Thevolume corresponds to the intensity of the chemiluminescence signalmultiplied by the surface area of the band.

5.2. Results

The Western blotting results are reproduced in Table 5, which gives thevolume of the bands corresponding to the tumor marker of interest forthe Western blotting analyses, as a function of the various samplestested. These results show that the tumor markers tested are indeedexpressed by the Caco-2 and HT-29 colon cancer lines, and also in thetissues, as shown with the extracts of tumor and mucosa, obtained fromthe patients. The intensity of the signal obtained with an antibody on asample can be compared to the signals obtained with the other samplesand the same antibody. The technique used makes it possible to confirmthe presence or absence of the marker in the tissue (non-remote sample)and the specificity of the antibodies with respect to the markers. Thistechnique was not used in this example in the remote samples because itwould not make it possible to come to a conclusion regarding thepresence or absence of the tumor marker in the remote samples, nor todetermine whether the concentration of said tumor marker is increased ordecreased in said samples. Furthermore, the experimental scheme useddoes not make it possible to compare the reactivity of one antibody withanother.

TABLE 5 Tumor Tumor Mucosal Tumor Mucosal Tumor marker and tissue tissuetissue tissue tissue antibody Caco-2 HT-29 GHBD001 GHBD004 GHBD004CLSP109 CLSP109 LEI 21B10A5 8365 7678 NT 60200 36506 NT NT 10E1H1 0 0 NT13357 6893 NT NT Ezrin 4A7A6C1 123436 116448 42480 15303 67439 NT NT4D9B9 121564 103833 4205 1810 11848 NT NT 4G11B5 105774 120944 6362623879 130522 NT NT 8B5G6G4 NT NT NT NT NT NT NT 4A9H5 7066 4742 NT NT NT1588 2446 5G2D12 1361 558 NT NT NT 206 259 8A8G6 458 199 NT NT NT 51 409D2A11 1580 1736 NT NT NT 542 476 Aminoacylase 1 2A7F6 10687 4787 NT NTNT 4477 7238 11H7D9 217664 232005 36093 10513 30233 NT NT I-plastin3D11D10 136725 NT NT NT NT 275477 246564 8C8C5 557 1110 4364 77 0 NT NTCalreticulin 5C10H10 2842 3040 NT NT NT 2503 3294 11B6D11 3261 2937 NTNT NT 2070 2764 LDH 3F11E11 45391 NT NT NT NT 30411 13942 12F10G8 122907154593 11841 15811 53285 NT NT Galectin-3 12F8A12 245712 65790 1826212961 7307 NT NT 14A5G1 254531 120010 79833 98361 45872 NT NT NT: nottested.

5.3. Monoclonal Antibodies Directed Against I-Plastin

In the patient GHBD004, the 8C8C5 antibody does not light up, or onlyvery weakly lights up, the band which corresponds to I-plastin. Thepresence of I-plastin in these samples can be demonstrated using, forexample, the 8G2D2 antibody, which has a better affinity for I-plastinin blotting.

Since I-plastin is a member of a family of proteins comprising 2 otherisoforms (L-plastin and T-plastin) with which it has more than 70%homology, we tested all the clones of monoclonal antibodies obtained,for their reactivity with respect to the GST-plastin-L and GST-plastin-Tproteins (provided by the Institut Curie). At the end of this screening,we selected the clones 3D11D10, 8C8C5, 3A3H2 and 8G2D2 which do notexhibit any cross-reactivity with the other members of the family. Theseantibodies are indeed specific for the I-plastin isoform.

6. Characterization of the Epitopes Recognized by the MonoclonalAntibodies Using the Spotscan Technique

6.1. Methodology

The Spotscan technique, adapted according to Frank and Döring⁷³, makesit possible to simultaneously synthesize a large number of peptidesattached to a cellulose membrane. These peptides reproduce the sequenceof the target antigen in the form of peptides of 8 to 12 amino acids,overlapping by 1 to 4 residues. These peptides are then brought intocontact with the antibody to be studied, in a colorimetric test of Blottype, and identification of the immunoreactive peptides makes itpossible to deduce the minimum sequence of the antibody epitope and toprecisely locate it on the antigen.

The synthesis is carried out on a cellulose membrane uniformly carryingarms of polyethylene glycol (PEG) of 8 to 10 units in length, having afree NH₂ function at the end of the chain. It takes place from theC-terminal end to the N-terminal end of the peptides. The amino functionof the amino acids is protected with an Fmoc(9-fluorenylmethyloxycarbonyl) group, and the side chains of said aminoacids, which are capable of reacting during the synthesis, are alsoprotected with trityl, t-butyl or t-butyl ether groups. The amino acidstock solutions are prepared at a concentration of 0.33 M in NMP(N-methylpyrrolidone) containing 0.5 M of HOBt (hydroxybenzotriazole).The amino acids are deposited using the ASP 222 automated device(Abimed, Langenfeld, Germany), controlled by means of the AutoSpot XLsoftware. The use of this automated device makes it possible tosimultaneously produce up to 4 membranes of 96 spots, i.e. 384 peptides.

For one amino acid coupling cycle, the automated device deposits 0.7 μlof the solution of extemporaneously activated amino acid (one volume of1.1 M diisopropylcarbodiimide solution diluted in NMP per 3 volumes ofamino acid stock solution) onto the membranes. This deposit is repeateda second time, and the membranes are then rinsed in DMF(N,N-dimethylformamide). The NH₂ groups which have not reacted are thenacetylated by 4 to 6 incubations for 10 minutes in a 10% solution ofacetic anhydride in DMF in order to prevent the appearance of abortiveor truncated peptides. After 3 washes for 2 minutes in DMF, the Fmocgroups protecting the amino function of the amino acids are cleaved byincubation for 5 minutes in a 20% solution of piperidine in DMF. After 4washes in DMF, the spots are colored using a 1% solution of bromophenolblue in DMF, and the membrane is then rinsed 3 times in methanol anddried in the open air before the next coupling cycle.

The protocol is repeated for the addition of each new amino acid. Afterthe coupling of the final amino acid, the peptides are acetylated inorder to make it possible to block all the free NH₂ groups, thuspreventing the addition of another amino acid. The side chains of allthe peptides are then deprotected by incubation of the membranes in atrifluoroacetic acid/dichloromethane/triisobutylsilane (5:5:0.3) bathfor 1 hour. The membranes are then rinsed 4 times in dichloromethane, 3times in DMF and 3 times in methanol, before being dried in the open airand stored at −20° C. until the immunorevealing.

In order to immunoreveal the spots with a monoclonal antibody, themembranes are first rinsed in methanol, and then washed in TBS (50 mMTris-HCl, pH 8.0, 140 mM NaCl, 3 mM KCl) before being incubatedovernight at ambient temperature in the saturating solution (10×concentrated, casein-based solution (Western Blocking reagent, Roche)diluted in TBS-0.05% Tween 20 (TBS-T) and containing 5% of sucrose).After washing for 10 minutes in TBS-T, the membranes are incubated for 1h30 at 37° C. with the monoclonal antibody diluted to 20 μg/ml insaturating solution. The membranes are subsequently washed 3 times inTBS-T, and are then incubated with the alkaline-phosphatase-coupledanti-mouse conjugate (Jackson Immunoresearch), diluted to 1/2000th insaturating solution. After 2 washes for 10 minutes in TBS-T, and then 2washes in CBS (10 mM citric acid, pH 7, 140 mM NaCl, 3 mM KCl), therevealing agent, prepared extemporaneously (600 μM,5-bromo-4-chloro-3-indoyl phosphate, 720 μM thiazolyl blue tetrazoliumbromide, and 5 mM MgCl₂ in CBS), is brought into contact with themembrane for 30 to 45 minutes, in the dark. The immunoreactive peptidesappear blue-violet in color. After 3 rinses in distilled water, themembranes are scanned and then stored in water until regeneration.

The regeneration makes it possible to eliminate the antibodies and theconjugates bound to the peptides, which thus makes it possible to carryout a further test for immunoreactivity with respect to anotherantibody. The membranes undergo a series of washes, each for 10 minutes:1 wash in distilled water, 6 washes in DMF, 3 washes in regeneratingbuffer A (8 M urea, 35 mM SDS (sodium dodecyl sulfate), 0.1%β-mercaptoethanol), 3 washes in regenerating buffer B (distilledwater/ethanol/acetic acid 4:5:1), then 2 washes in methanol. Themembranes are then dried in the open air before being stored at −20° C.

6.2. Results

Table 6 reproduces the epitopes recognized by the 8 antibodies studiedusing the Spotscan technique.

TABLE 6 Antibody Epitope No. Sequence of the epitope^(a) (SEQ ID No.)4A7A6C1 1 LSEQIQRALQLE (365-376) (SEQ ID No. 1) 4D9B9 2 LAAELAEYT(417-425) (SEQ ID No. 2) 4G11B5 1 LSEQIQRALQLE (365-376) (SEQ ID No. 1)8B5G6G4 3 QIQRALQLEEER (368-379) (SEQ ID No. 3) 4A9H5 4 DYEEKTKK(353-360) + QAKFGD (131-136) (SEQ ID No. 4) + (SEQ ID No. 5) 5G2D12 2LAAELAEYT (417-425) (SEQ ID No. 2) 8A8G6 5 LEADRMAAL (389-397)+ LEEARRRKED (431-440) (SEQ ID No. 6) + (SEQ ID No. 7) 9D2A11 6LEADRMAALRAK (389-400) (SEQ ID No. 8) ^(a)Amino acid sequence of theregion of binding of Ezrin to the antibody tested. The numbers betweenparentheses correspond to the position of the epitope on the Ezrin aminoacid sequence, the numbering beginning at the initial methionine.

The 8 anti-Ezrin monoclonal antibodies recognize 6 epitopes of theprotein, which were numbered from 1 to 6 (Table 6). The 4A7A6C1 and4G11B5 antibodies have the same epitope (epitope n° 1). The 4D9B9H6 and5G2D12 antibodies also have the same epitope (epitope n° 2). The 8A8G6and 9D2A11 antibodies have partially overlapping, but not completelyidentical, epitopes.

7. Detection of Ezrin by Sandwich ELISA

7.1. Methodology

Ezrin was detected by sandwich immunoassay using, for example, theVidas® ELISA automated device (bioMérieux). To do this, the ELISA assaywas constructed using the reagents of the Vidas® HBs Ag Ultra kit(bioMérieux, Cat. No. 30315). The reagents were used as described in thecorresponding information sheet (ref. 11728 D-FR-2005/05), modified inthe following way:

1. The cones were sensitized with the capture antibodies to be tested(4A9H5, 5G2D12, 8A8G6) at a concentration of 30 μg/ml.

2. The content of the second well of the HBs Ag Ultra cartridge wasreplaced with 300 μl of revealing antibody to be tested (4A7A6C1 and4G11B5) coupled to biotin, diluted to 1 μg/ml in the buffer of thesecond well of the Vidas® HBs Ag Ultra kit (buffer with goat serum andsodium azide at 1 g/l).

3. The Caco2 lysate (containing ezrin in native form), prepared bysonicating the cell pellet in PBS (50 μl), is diluted directly in thesecond well of the HBs Ag Ultra cartridge.

4. The ELISA reaction was carried out using the Vidas® automated deviceand the HBs Ag Ultra protocol, of which the step of incubating thesample with the capture and revealing antibodies had been brought to 100cycles.

5. The results were obtained in the form of crude values aftersubtraction of the background noise (reading of the substrate beforereaction). The signal is in RFV, relative fluorescence value.

7.2. Results

Table 7 reproduces the reactivity of the various combinations ofantibodies on a 1/50 dilution of the Caco2 lysate. The 2 antibodieswhich bind to epitope No. 1, 4A7A6C1 and 4G11B5, generate identicalsignal profiles. It is possible to detect Ezrin using the combination ofepitopes Nos 1 and 4, or Nos 1 and 2, or else Nos 1 and 5. When there isno reactivity with the tumor marker, the signals observed are muchlower, of the order of 2.5 to 3 RFV. The combination of epitopes Nos 1and 4 makes it possible to obtain the highest signals.

TABLE 7^(a) Detection antibody (epitope n^(o)1) Capture antibody4A7A6C1biotin 4G11B5biotin 4A9H5 (epitope n^(o)4) 6229.5 6166 5G2D12(epitope n^(o)2) 52.5 46.5 8A8G6 (epitope n^(o)5) 241.5 234 Negativecontrol 3 2.5 ^(a)Vidas sandwich immunoassay signal, in RFV (relativefluorescence unit).

EXAMPLE 3 Serum Assays for the Tumor Markers

1. Patients and Specimens

Blood samples are collected from a network of 8 clinical centersdistributed throughout France, in the context of 2 Huriet-law protocols.

In order to obtain serum, the blood sample is taken on a dry tube. Inorder to obtain plasma, the blood sample is taken on an EDTA tube. Aftercoagulation, the tube is centrifuged for 10 min at 1000 g, and the serumis removed, aliquoted and stored at −80° C. The tube of plasma isdirectly centrifuged for 10 min at 1000 g, and the plasma is removed,aliquoted and stored at −80° C. The samples are completely documentedfor the clinical history of the patients.

2. Serum Assay for the LEI Tumor Marker

The LEI protein was assayed using the antibodies described in Example 2and an ELISA assay using the Vidas® automated device (bioMérieux). To dothis, the ELISA assay was constructed using the reagents of the Vidas®HBs Ag Ultra kit (bioMérieux, Cat. No. 30315). The reagents were used asdescribed in the corresponding information sheet (ref 11728D-FR-2005/05), with the following modifications:

-   -   1. The cones were sensitized with the capture antibody 10E1H1 at        a concentration of 10 μg/ml.    -   2. The content of the second well of the HBs Ag Ultra cartridge        was replaced with 300 μl of revealing antibody 21B10A5, coupled        to biotin, diluted to 1 μg/ml in the buffer of the second well        of the Vidas® HBs Ag Ultra kit (buffer with goat serum and        sodium azide at 1 g/l).    -   3. The serum, plasma or stool samples (50 μl) were diluted        directly in the second well of the HBs Ag Ultra cartridge, pure        or after a prior dilution to 1/20 in the buffer of the second        well of the Vidas® HBs Ag Ultra kit (buffer with goat serum and        sodium azide at 1 g/l).    -   4. The ELISA reaction was carried out using the Vidas® automated        device and the protocol of the HBs Ag Ultra kit.    -   5. The results were obtained in the form of crude values after        subtraction of the background noise (reading of the substrate        before reaction).        A standard curve was established by assaying a range of        concentrations of the tumor marker in the form of recombinant        protein. The standard curve was plotted by reporting the        concentration of the tumor marker along the x-axis and the        signal read by Vidas® (RFV or Relative Fluorescence Value) along        the y-axis. The concentration of tumor marker present in the        body fluid to be assayed (blood, serum, plasma, stool) was        calculated by reporting the concentration corresponding to the        RFV signal read by Vidas®.

The amounts obtained for the patients analyzed are reported in FIG. 1.It may be noted, on this figure, that 3 sera of patients having stage IVcolorectal cancer and 1 serum of a patient having stage III colorectalcancer show a clear increase in their amount of serum LEI.

3. Serum Assay for the Ezrin Tumor Marker

The Ezrin protein was assayed using the antibodies described in detailin Example 2 and an ELISA assay using the Vidas® automated device(bioMérieux). To do this, the ELISA assay was constructed using thereagents of the Vidas® HBs Ag Ultra kit (bioMérieux, Cat. No. 30315).The reagents were used as described in the corresponding informationsheet (ref. 11728 D-FR-2005/05), with the following modifications:

1. The cones were sensitized with the capture antibody 4A9H5 at aconcentration of 30 μg/ml.

2. The content of the second well of the HBs Ag Ultra cartridge wasreplaced with 300 μl of revealing antibody 4A7A6C1, coupled to biotin,diluted to 1 μg/ml in the buffer of the second well of the Vidas® HBs AgUltra kit (buffer with goat serum and sodium azide at 1 g/l).

3. The serum, plasma and stool samples (50 μl) were diluted directly inthe second well of the HBs Ag Ultra cartridge.

4. The ELISA reaction was carried out using the Vidas® automated deviceand the HBs Ag Ultra protocol, in which the step of incubating thesample with the capture and revealing antibodies had been taken to 100cycles.

5. The results were obtained in the form of crude values aftersubtraction of the background noise (reading of the substrate beforereaction).

The concentration of the tumor marker present in the body fluid to beassayed (blood, serum, plasma, stool) was calculated according to theprocedure described in paragraph 2 regarding the assaying of LEI.

The results of the assay for serum Ezrin in the patients by ELISA on aVidas automated device are given in Table 8.

TABLE 8 Pathological Serum sample Ezrin Condition^(a) identifier NatureStage TNM^(b) ng/ml CRC+ CLSP047/F0 Serum I T1N0M0 9 CRC+ CLSP076/F0Serum II T3N0M0 8.579 CRC+ GHBD020/F0 Serum II T3N0M0 21.362 CRC+GHBD023/F0 Serum II T3N0M0 7.955 CRC+ GHBD025/F0 Serum II T3N0M0 5.985CRC+ GHBD029/F0 Serum II T3N0M0 13.343 CRC+ CBSE005/F0 Plasma III T3N1M020.248 CRC+ CLSP050/F0 Plasma III T2N1M0 11.706 CRC+ CLSP072/F0 SerumIII T3N1M0 9.252 CRC+ CLSP089/F0 Serum III T4N2M0 8.916 CRC+ CLSP090/F0Serum III T1N1M0 18.460 CRC+ GHBD019/F0 Serum III T3N1M0 7.810 CRC+CBSE008/F0 Serum IV T4N3M1 9.781 CRC+ CBSE021/F0 Serum IV T4N2M1 18.846CRC+ CBSE026/F0 Serum IV T4N1M1 52.641 CRC+ CLSP068/F0 Serum IV TxNxM1392.369 CRC+ CLSP156/F0 Serum IV T4N0M1 12.188 CRC+ CSEM005/F1 Serum IVT4N2M1 89.383 CRC+ CLSP153/F0 Serum III/IV T3N2Mx 12.477 CRC− N017197/F0Plasma 12.958 CRC− N017250/F0 Serum 8.435 CRC− N017349/F0 Serum 19.668CRC− N018640/F0 Serum 8.964 CRC− N030068/F0 Serum 11.802 CRC− N143574/F0Serum 17.155 CRC− N146628/F0 Serum 12.091 CRC− N148324/F0 Serum 15.900CRC− N314199/F0 Serum 5.314 CRC− N314324/F0 Serum 13.006 CRC− N417852/F0Serum 26.598 CRC− N491079/F0 Plasma 5.842 CRC− N491124/F0 Plasma 8.531CRC− N491722/F0 Serum 23.735 CRC− N518569/F0 Serum 14.453 CRC−N835827/F0 Plasma 13.151 CRC− N836221/F0 Plasma 11.802 CRC− N836301/F0Plasma 10.454 CRC− N744056/F0 Serum 20.974 CRC− N835886/F0 Serum 14.887CRC− N835931/F0 Serum 10.358 CRC− N836125/F0 Serum 22.911 ^(a)CRC+:patients having colorectal cancer/CRC−: healthy individual ^(b)TNM:stage of tissue invasion (T), lymph node invasion (N) and remoteinvasion (metastases, M)

The amounts obtained for the patients analyzed are reported in FIG. 2.It may be noted, in this figure, that 3 sera from patients having stageIV colorectal cancer show a clear increase in their amount of serumEzrin.

4. Serum Assay for the Aminoacylase 1 Tumor Marker

The Aminoacylase 1 protein was assayed using the antibodies described inExample 2 and an ELISA assay using the Vidas® automated device(bioMérieux). To do this, the ELISA assay was constructed using thereagents of the Vidas® HBs Ag Ultra kit (bioMérieux, Cat. No. 30315).The reagents were used as described in the corresponding informationsheet (ref. 11728 D-FR-2005/05), with the following modifications:

-   -   1. The cones were sensitized with the capture antibody 2A7F6 at        a concentration of 20 μg/ml.    -   2. The content of the second well of the HBs Ag Ultra cartridge        was replaced with 300 μl of revealing antibody 11H7D9, coupled        to biotin, diluted to 1 μg/ml in the buffer of the second well        of the Vidas® HBs Ag Ultra kit (buffer with goat serum and        sodium azide at 1 g/l).    -   3. The serum, plasma or stool samples (100 μl) were diluted        directly in the second well of the HBs Ag Ultra cartridge.    -   4. The ELISA reaction was carried out using the Vidas® automated        device and the HBs Ag Ultra protocol, in which the step of        incubating the sample with the capture and revealing antibodies        had been taken to 100 cycles.    -   5. The results were obtained in the form of crude values after        subtraction of the background noise (reading of the substrate        before reaction).

The concentration of the tumor marker present in the body fluid to beassayed (blood, serum, plasma, stool) was calculated according to theprocedure described in paragraph 2 regarding the assaying of LEI.

The amounts obtained for the patients analyzed are reported in FIG. 3.It may be noted, in this figure, that 1 serum from a patient havingstage II colorectal cancer, 1 serum from a patient having stage IIIcolorectal cancer and 2 sera from patients having stage IV colorectalcancer show a clear increase in their amount of serum Aminoacylase 1.

5. Serum Assay for the L-FABP Tumor Marker

We used an ELISA kit marketed by the company Hycult Biotechnology toassay the human L-FABP protein (Cat. No. HK404). This kit makes itpossible to quantify the L-FABP protein in cell culture supernatants orin serum, plasma or urine, in order to determine the presence of lesionsin the liver. We followed the procedure recommended by the manufacturer,with 2 modifications: the incubations were carried out at 37° C. and notat ambient temperature, the sera were diluted to 1/10^(th) before theassay. The assaying of the L-FABP protein can be carried out byalternative techniques, well known to those skilled in the art.

FIG. 4 gives the results of this assay. In the serum panel that wetested, 41 patients out of 141 having colorectal cancer have a serumL-FABP concentration of greater than 17 ng/ml, whereas, in the controlgroup, no individual exceeds this value. Among these 41 patients, are 8patients with stage I colorectal cancer, 8 with stage II colorectalcancer, 13 with stage III colorectal cancer and 12 with stage IVcolorectal cancer. The mean serum L-FABP concentration observed for 141patients with colorectal cancer is 16.6±1.3 ng/ml. The mean value is6.6±0.2 ng/ml for 112 healthy individuals (negative controls). Thisdifference is statistically significant (P<0.0001, one-sided t-test withWelch's correction for unequal variances).

6. Serum Assay for the I-FABP Tumor Marker

We used an ELISA kit marketed by the company Hycult Biotechnology toassay the human I-FABP protein (Cat. No. HK406). This kit makes itpossible to quantify the I-FABP protein in cell culture supernatants orin serum, plasma or urine, in order to determine the presence ofischemic lesions in the small intestine. We followed the procedurerecommended by the manufacturer. The assaying of the I-FABP protein canbe carried out by alternative techniques, well known to those skilled inthe art.

FIG. 5 gives the results of this assay. In the serum panel that wetested, 15 patients out of 40 having colorectal cancer have a serumI-FABP concentration of greater than 40 pg/ml, whereas, in the controlgroup, only 2 individuals out of 24 exceed this value. More clearly, 3sera of patients having stage I colorectal cancer, 2 sera of patientshaving stage III colorectal cancer and 1 serum of a patient having stageIV colorectal cancer have a serum I-FABP concentration of greater than100 pg/ml. No concentration above this value was found in the CRC−control group.

7. Serum Assay for the Apolipoprotein AI Tumor Marker

The assaying of serum Apolipoprotein AI was carried out by means of twodifferent immunoassay techniques. Firstly, we used a microplate sandwichELISA. The 96-well plates were coated with the anti-Apo AI monoclonalantibody, clone 1404 (Biodesign Cat. No. H45404) at 1 μg per well. After3 washes with PBS-0.05% Tween 20 (PBS-T), the plates were saturated with10% milk in PBS-T for 1 h at 37° C. The plates were washed a further 3times in PBS-T, 100 μl of the dilutions of the standard range or 100 μlof the 1/100 000 dilution of the test serum samples were deposited ontothe plates, and the plates were incubated for 2 h at 37° C. The standardrange was prepared by diluting the Apo AI protein (Biodesign Cat. No.A50620H) in PBS-T, BSA 1% (1.6 to 100 ng/ml). After 3 washes with PBS-T,the polyclonal detection antibody coupled to horseradish peroxidase(Biodesign Cat. No. K45452P) was added at 0.1 μg per well, and theplates were incubated for 2 h at 37° C. A further 3 washes with PBS-Twere carried out, before adding the OptEIA substrate (BD), at 100μl/well. After 20 min, when the development of the color had takenplace, the reaction was stopped with 2N sulfuric acid and the absorbenceat 450 nm was measured.

FIG. 6A gives the results of this assay. We demonstrated a decrease inserum concentration of Apo AI in individuals with colorectal cancer. Themean concentration in 38 individuals with stage I to IV CRC is 675±36μg/ml, whereas it is much higher in 27 healthy individuals (controls):1040±39 μg/ml. This difference is statistically very significant(P<0.0001, one-sided t-test). By way of comparison, in 13 individualswith liver cancer, the mean serum concentration of Apo AI is 1175±87μg/ml with the sandwich ELISA technique used. The decrease in the serumconcentration demonstrates that Apo AI is therefore a specific marker ofcolorectal cancer, it being possible for this decrease to bedemonstrated by means of an immunoassay.

The second assaying technique that was used is a multiplex assaymarketed by the company Linco, which makes it possible to assay severalApolipoproteins, including AI and AII, simultaneously, in the samesample (Cat. No. APO-62K). The procedure recommended by the manufacturerwas applied.

FIG. 6B gives the results of this assay. The decrease in the serumconcentration of Apo AI in patients with CRC is confirmed with thissecond technique. The mean concentration of Apo AI in 34 individualswith stage I to IV CRC is 768±30 μg/ml, whereas it is much higher in 17healthy individuals (controls): 1194±51 μg/ml. This difference isstatistically very significant (P<0.0001, one-sided t-test).

8. Serum Assay for the Apolipoprotein AII Tumor Marker

The assaying of serum Apolipoprotein AII was carried out with the Lincomultiplex kit. FIG. 7 gives the results of this assay. We demonstrated adecrease in the serum concentration of Apo AII in the individuals withcolorectal cancer. The mean concentration of Apo AII in 34 individualswith stage I to IV CRC is 170±11 μg/ml, whereas it is much higher in 17healthy individuals (controls): 277±16 μg/ml. This difference isstatistically very significant (P<0.0001, one-sided t-test).

9. Serum Assay for the I-Plastin Tumor Marker

The I-Plastin protein was assayed using the antibodies described inExample 2 and an ELISA assay using the Vidas® automated device(bioMérieux). To do this, the ELISA assay was constructed using thereagents of the Vidas® HBs Ag Ultra kit (bioMérieux, Cat. No. 30315).The reagents were used as described in the corresponding informationsheet (ref. 11728 D-FR-2005/05), with the following modifications:

-   -   1. The cones were sensitized with the capture antibody 3D11D10        at a concentration of 15 μg/ml.    -   2. The content of the second well of the HBs Ag Ultra cartridge        was replaced with 300 μl of revealing antibody 8C8C5, coupled to        biotin, diluted to 1 μg/ml in the buffer of the second well of        the Vidas® HBs Ag Ultra kit (buffer with goat serum and sodium        azide at 1 g/l).    -   3. The serum, plasma or stool samples (100 μl) were diluted        directly in the second well of the HBs Ag Ultra cartridge.    -   4. The ELISA reaction was carried out using the Vidas® automated        device and the HBs Ag Ultra protocol.    -   5. The results were obtained in the form of crude values after        subtraction of the background noise (reading of the substrate        before reaction).

The concentration of the tumor marker present in the body fluid to beassayed (blood, serum, plasma, stool) was calculated according to theprocedure described in paragraph 2 regarding the assaying of LEI.

The amounts obtained for the patients analyzed are reported in FIG. 8.The 2 sera of patients having colorectal cancer who were tested show aclear increase in their amount of serum I-Plastin.

10. Serum Assay for the Group-B Tumor Markers

The Beta2-Microglobulin, CEA, CA19-9 and Testosterone tumor markers wereassayed using the assay kits of the applicant, respectively Vidas®β2-Microglobulin, Vidas® CEA, Vidas® CA19-9™ and Vidas® Testosterone,according to the procedure specific to each kit.

The E-Cadherin protein was assayed using the E-Cadherin EIA kit (TakaraBiochemicals, Tokyo, Japan) according to the procedure of the kit.

The Regenerating Islet-Derived Protein 3 Alpha protein, otherwise knownas pancreatitis associated protein (PAP1), was assayed using thePANCREPAP ELISA kit (DynaBio, Marseille, France) according to theprocedure of the kit.

The Galectin-3 and LDH proteins were assayed using the antibodiesdescribed in Example 2. The Proteasome 20 S was assayed using theantibodies described in patent EP 0434670. To do this, the ELISA assayswere constructed using the Vidas® automated device (bioMérieux) and thereagents of the Vidas® HBs Ag Ultra kit (bioMérieux, Cat. No. 30315).The reagents were used as described in the corresponding informationsheet (ref. 11728 D-FR-2005/05), with the following modifications:

-   -   1. The cones were sensitized with the capture antibody at a        concentration of between 5 and 30 μg/ml.    -   2. The content of the second well of the HBs Ag Ultra cartridge        was replaced with 300 μl of revealing antibody, coupled to        biotin, diluted to 1 μg/ml in buffer with goat serum and sodium        azide at 1 g/l.    -   3. The serum, plasma or stool samples were diluted directly in        the second well of the HBs Ag Ultra cartridge after, if        necessary, a dilution in buffer of the second well.    -   4. The ELISA reaction was carried out using the Vidas® automated        device and the HBs Ag Ultra protocol. The step of incubating the        sample with the capture and revealing antibodies was between 14        and 100 cycles.    -   5. The results were obtained in the form of crude values after        subtraction of the background noise (reading of the substrate        before reaction).

The concentration of the tumor marker present in the body fluid to beassayed (blood, serum, plasma, stool) was calculated according to theprocedure described in paragraph 2 regarding the assaying of LEI. Theassay conditions for various tumor markers have been reproduced in Table7.

TABLE 9 Protein Galectin-3 LDH-B Proteasome 20 S Capture antibody12F8A12 at CC3F11E11 at GD6 at 30 μg/mL 15 μg/mL 10 μg/mL Revealingantibody 14A5G1 12F10G8 7A11 Goat serum in dilution with with withoutbuffer Stool volume  50 μL 50 μL 200 μL Serum volume  50 μL 50 μL 100 μLSample deposit 2^(nd) well 2^(nd) well 1^(st) well Incubation time 100cycles 14 cycles  14 cycles

The amounts obtained for the patients analyzed with thebeta2-Microglobulin, CEA, CA19-9, Testosterone, E-Cadherin, RegeneratingIslet-Derived Protein 3 Alpha, Galectin-3, LDH and Proteasome 20S tumormarkers have been reported respectively in FIGS. 9 to 17.

Three sera of patients having colorectal cancer show an increase intheir amount of serum β2-Microglobulin.

Ten sera of patients having colorectal cancer show an increase in theiramount of serum CEA. More clearly, 1 serum of a patient having stage IIIcolorectal cancer and 7 sera of patients having stage IV colorectalcancer show a considerable increase in their amount of serum CEA.

Nine sera of patients having colorectal cancer show an increase in theiramount of serum CA 19-9. More clearly, 1 serum of a patient having stageIII colorectal cancer and 7 sera of patients having stage IV colorectalcancer show a considerable increase in their amount of serum CA 19-9.

Ten sera of patients having colorectal cancer show a decrease in theiramount of serum Testosterone. More clearly, 1 serum of a patient havingstage II colorectal cancer, 1 serum of a patient having stage IIIcolorectal cancer and 2 sera of patients having stage IV colorectalcancer show a fall in their amount of serum Testosterone.

Two sera of patients having colorectal cancer show an increase in theiramount of serum Regenerating Islet-Derived Protein 3 Alpha.

Four sera of patients having stage IV colorectal cancer, 2 sera ofpatients having stage III colorectal cancer and 1 serum of a patienthaving stage II colorectal cancer show a clear increase in their amountof serum Galectin-3.

EXAMPLE 4 Use of the Serum Assays for Tumor Markers in Combination

The applicant showed in Example 3 that abnormally elevated or abnormallyreduced amounts of tumor markers could be observed in the bloodstream ofcertain patients having colorectal cancer. Surprisingly, the increase orthe decrease in the amount, in the blood, of two given markers is notsystematically observed in the same patients. As a result, thecombination of several tumor markers makes it possible to increase thenumber of patients identified as having colorectal cancer. Thus, apatient A may present an increase or a decrease in one or more tumormarkers (group X), it being possible for said markers of group X to benormal in a patient B; in this same patient B, one or more other tumormarkers (group Y) may be elevated or reduced, it being possible for saidmarkers of group Y to be normal in patient A.

The various tumor markers assayed by the applicant may thus be combinedby means of various mathematical algorithms well known to those skilledin the art. By way of illustration, and without this example beingexhaustive in nature, the following method was carried out:

-   -   1. A threshold value was set for each tumor marker.    -   2. When the amount of the tumor marker in the blood was        increased in the case of colorectal cancer, the amount in the        blood, obtained for a given patient, was divided by its        threshold value. When the amount of the tumor marker in the        blood was decreased in the case of colorectal cancer, the amount        in the blood, obtained for a given patient, was inverted and        then multiplied by its threshold value.    -   3. When the “amount in the blood divided by threshold value”        ratio was greater than 1, the ratio was multiplied by a        coefficient, for example 10. The value thus obtained was named        the “score”, for the patient studied, for the tumor marker under        consideration.    -   4. The scores obtained for various tumor markers were added,        with them being weighted by a factor specific to each marker. In        the case of the example below, all the weighting factors were        set at 1.    -   5. The sum of the scores was divided by the total number of        scores added, and the value thus obtained was named the “total        score”.    -   6. The patient is diagnosed as having colorectal cancer when his        or her total score is increased relative to a threshold score.

The total scores for a selection of 2, 4 and 8 markers comprising Ezrinare given in Table 10.

The combination of the Ezrin and Aminoacylase 1 tumor markers thus makesit possible to obtain, for the same group of 19 patients, increasedtotal scores “2” in 6 patients having colorectal cancer, whereasassaying Ezrin or Aminoacylase 1 alone showed an increase, respectively,in 3 and 4 patients only.

The combination of the Ezrin, Beta2-Microglobulin, Aminoacylase 1 andCEA tumor markers thus makes it possible to obtain, for the same groupof 19 patients, increased total scores “4” in 15 patients havingcolorectal cancer, whereas assaying Ezrin, Beta2-Microglobulin,Aminoacylase 1 or CEA alone showed an increase, respectively, in 3, 4, 3and 9 patients only.

The combination of the Ezrin, Beta2-Microglobulin, Aminoacylase 1,Proteasome 20S, L-FABP, PAP, E-Cadherin and CEA tumor markers thus makesit possible to obtain, for the same group of 19 patients havingcolorectal cancer, increased total scores “8” in 17 patients, whereasassaying Ezrin, Beta2-Microglobulin, Aminoacylase 1, Proteasome 20S,L-FABP, PAP, E-Cadherin or CEA alone showed an increase, respectively,in 3, 4, 3, 6, 5, 3, 1 and 9 patients only.

TABLE 10 Total Total Total Pathological Sample Ezrin score score scorecondition identifier Stage TNM ng/ml 2^(a) 4^(b) 8^(c) CRC+ CLSP047/F0 IT1N0M0 9.06 0.27 0.47 0.33 CRC+ CLSP076/F0 II T3N0M0 8.58 0.28 0.45 1.95CRC+ GHBD020/F0 II T3N0M0 21.36 0.57 0.57 1.83 CRC+ GHBD023/F0 II T3N0M07.95 0.24 2.85 1.62 CRC+ GHBD025/F0 II T3N0M0 5.99 0.23 3.33 1.91 CRC+GHBD029/F0 II T3N0M0 13.34 9.52 4.96 2.73 CRC+ CBSE005/F0 III T3N1M020.25 121.20 60.62 42.13 CRC+ CLSP050/F0 III T2N1M0 11.71 0.36 4.11 3.52CRC+ CLSP072/F0 III T3N1M0 9.25 0.17 7.79 3.99 CRC+ CLSP089/F0 IIIT4N2M0 8.92 0.32 21.60 11.11 CRC+ CLSP090/F0 III T1N1M0 18.46 0.45 0.360.36 CRC+ GHBD019/F0 III T3N1M0 7.81 0.25 4.87 2.69 CRC+ CBSE008/F0 IVT4N3M1 9.78 27.55 13.86 7.02 CRC+ CBSE021/F0 IV T4N2M1 18.85 0.47 162.8583.63 CRC+ CBSE026/F0 IV T4N1M1 52.64 10.02 135.24 72.28 CRC+ CLSP068/F0IV TxNxM1 392.37 74.03 261.23 142.23 CRC+ CLSP156/F0 IV T4N0M1 12.190.39 30.46 17.03 CRC+ CSEM005/F1 IV T4N2M1 89.38 16.80 8.60 7.07 CRC+CLSP153/F0 III/IV T3N2Mx 12.48 0.35 13.40 8.91 CRC− N017197/F0 13.0 0.240.27 0.18 CRC− N017250/F0 8.4 0.16 0.14 0.24 CRC− N017349/F0 19.7 0.370.18 0.22 CRC− N018640/F0 9.0 0.17 0.14 0.23 CRC− N030068/F0 11.8 0.310.36 0.35 CRC− N143574/F0 17.2 0.39 0.62 0.51 CRC− N146628/F0 12.1 0.320.49 0.47 CRC− N148324/F0 15.9 0.37 0.48 0.51 CRC− N314199/F0 5.3 0.190.24 0.33 CRC− N314324/F0 13.0 0.38 0.25 0.48 CRC− N417852/F0 26.6 0.600.41 0.49 CRC− N491079/F0 5.8 0.40 0.25 0.34 CRC− N491124/F0 8.5 0.160.19 0.15 CRC− N491722/F0 23.7 0.95 0.53 0.43 CRC− N518569/F0 14.5 0.370.26 0.48 CRC− N835827/F0 13.2 0.33 0.41 0.38 CRC− N836221/F0 11.8 0.290.46 0.38 CRC− N836301/F0 10.5 0.27 0.52 0.61 CRC− N744056/F0 21.0 0.500.25 0.35 CRC− N835886/F0 14.9 0.36 0.18 0.33 CRC− N835931/F0 10.4 0.300.15 0.30 CRC− N836125/F0 22.9 0.52 0.26 0.30 Threshold 26.60 0.95 0.620.61 ^(a)combination of Ezrin and Aminoacylase 1 ^(b)combination ofEzrin, Beta2-Microglobulin, Aminoacylase 1 and CEA ^(c)combination ofEzrin, Beta2-Microglobulin, Aminoacylase 1, Proteasome 20S, L-FABP, PAP,E-Cadherin and CEA

EXAMPLE 5 Fecal Tumor Marker Assays

The stools are extracted using a piece weighing approximately 1 g, towhich 10 ml of 100 mM sodium phosphate buffer, pH 7.2, containing 1 g/Lof azide are added. The mixture is homogenized on a vortex for 1 min.The sample is then subjected to 4 cycles of ultrasound for 7 s on ice.The unsolubilized fraction is removed by centrifugation at 2000 g, for10 min at 4° C. The supernant is stored at +30° C. until it is assayed.

The ELISA assays described in Example 3 were used to search for thetumor markers in the stools after, if necessary, an appropriate dilutionof the stools in the buffer of the first well of the HBs Ag Ultracartridge.

The assay determinations with the tests, Aminoacylase 1, Galectin-3 andProteasome 20S, have been represented, respectively, in FIGS. 18 to 20.An increase in the amount of Aminoacylase 1, of Galectin-3 and ofProteasome 20S is observed, respectively, for 10, 14 and 8 stools ofpatients having colorectal cancer.

EXAMPLE 6 Detection of the Tumor Markers by the ELISPOT Technique

1. Cell Culture

The LnCAP prostate cancer line is cultured in RPMI 1640 mediumsupplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvateand 10% FCS (all Gibco). The cells are used as a negative control.

The Caco-2 colorectal cancer line is cultured in DMEM medium containing2 mM L-glutamine, without FCS (all Gibco).

The HT-29 colorectal cancer line is cultured in MEM medium containing 2mM L-glutamine and 10% FCS (all Gibco).

The HT-29/B6 colorectal cancer line is cultured in DMEM mediumcontaining 4 mM L-glutamine, without FCS (all Gibco).

The cells are maintained at 37° C., in an incubator with 5% CO₂.

2. The ELISPOT Technique

This procedure makes it possible to determine the number of cellssecreting the protein. The 96-well ELISPOT plates with PVDF membranes(Multiscreen IP, Millipore) are coated with the mouse anti-tumor markermonoclonal antibody at 10 μg/ml (capture antibody, see Table 11 below,which gives the antibodies used in ELISPOT), 100 μl per well, in sterilePBS, overnight at +4° C. The plates are then washed with PBS andsaturated with culture medium containing 10% FCS. In parallel, the cellsare trypsinized, counted, and then diluted to 10⁵ cells/ml. 200 μl ofthis cell suspension are distributed per well, as are cascade dilutionsof this stock solution. The plates are then incubated for 20 h at 37° C.in a humid atmosphere at 5% CO₂, and then washed with PBS containing0.05% Tween-20. The remaining cells are then lyzed by treatment withice-cold water for 10 minutes, and then the plates are again washed. Therevealing antibody, the biotinylated monoclonal directed against thetumor marker to be assayed (Table 11), is then added at 0.1 μg/well(incubation for 2 h at ambient temperature). The spots are revealed byadding extravidin-alkaline phosphatase (Sigma) and the substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT,Biorad). The background noise corresponds to the number of spotsmeasured in the LnCap wells and varies between 0 and 8 spots under thereading conditions used. The average number of nonspecific spots wassubtracted from the specific signal.

TABLE 11 Marker Capture Ab Detection Ab LEI 10E1H1 21B10A5 Ezrin 4A9H54A7A6C1 Galectin-3 12F8A12 14A5G1

3. Results

The number of Caco-2, HT-29 and HT-29/B6 cells secreting the tumormarker of interest, per million incubated cells, is shown in FIG. 21.The ELISPOT technique makes it possible to confirm the release or thesecretion of the tumor markers by the colon cancer lines. It will bepossible to carry out a search for circulating tumor cells in patientsusing this technique, according to the method of patent application WO03/076942 filed by the applicant.

EXAMPLE 7 Immunohistochemical Detection of the Tumor Markers UsingColonic Tissues

1. Methodology

Firstly, the tissue-microarray slides are deparaffinized. For this, theyare incubated successively in the following baths for 10 minutes:methylcyclohexane (twice), 100% ethanol, 95% ethanol, 70% ethanol andwater. The slides are then rinsed with TBS containing 0.1% Tween 20(TBS-T), for 10 min, with stirring. The antigens are reactivated in 10mM citrate buffer, pH 6, by heating to 90° C. for 40 min, and then byallowing to cool to ambient temperature for 30 min. The endogenousperoxidases are inhibited by incubation in TBS-T containing 3% H₂O₂, for5 min. The slides are then saturated with 3% BSA in TBS-T, for 1 h at37° C., in a humid chamber.

The slides are then incubated for 2 h with the anti-Leukocyte ElastaseInhibitor (clone 3D9C2) or anti-1-Plastin (clone 8D6A3) primary antibodydiluted to 10 μg/ml in TBS-T containing 3% BSA (incubation at 37° C. ina humid chamber). After 3 washes of 10 min in TBS-T, the slides areincubated for 2 h at 37° C., in a humid chamber, with the horseradishperoxidase-coupled anti-mouse secondary antibody (Cat. No. 115-035-003Jackson Immunoresearch) diluted to 1/400 in the saturating solution. Theslides are washed 3 times for 10 minutes in TBS-T, and then 3 times for10 min in PBS. The slides are developed with the Sigma Fast substrate(Cat. No. D-4168, Sigma-Aldrich) for 5 min. The staining is stopped bywashing in PBS. Counterstaining with Harris hematoxylin (Cat. No. MHS16,Sigma-Aldrich) is carried out for 30 sec. After washing with water andwith PBS, the slides are mounted for observation under a microscope.

The antibodies used for the immunohistochemical labeling were selectedspecifically for this application, independently of their reactivity inELISA or in Western blotting.

2. Immunohistochemical Detection of Leukocyte Elastase Inhibitor

Tissue-microarray slides were used to screen a large number of samples.These samples are colonic tissues spotted onto slides. Thecharacteristics of the patients (characteristics of the colonic tissuespots present on the colorectal cancer tissue-microarray), and also theresults of the immunolabelings with the anti-Leukocyte ElastaseInhibitor antibody, are reproduced in Table 12.

TABLE 12 Labeling in Labeling Histology and genetic epithelial in theDiagnosis characterization cells stroma Malignant tumor Conservedadenocarcinoma Positive Negative Malignant tumor Conservedadenocarcinoma Positive Negative Normal Normal mucosa Negative NegativeNormal Normal mucosa Negative Negative Normal Normal mucosa NegativeNegative Normal Normal mucosa Negative Negative Benign tumor AdenomaNegative Negative Malignant tumor Conserved adenocarcinoma PositiveNegative Malignant tumor LOH adenocarcinoma Positive Negative Malignanttumor LOH adenocarcinoma Positive Negative Normal Normal mucosa NegativeNegative Normal Normal mucosa Negative Negative Normal Normal mucosaNegative Negative Normal Normal mucosa Negative Negative Malignant tumorLOH adenocarcinoma Negative Negative Malignant tumor LOH adenocarcinomaPositive Negative Malignant tumor MSI-high adenocarcinoma PositiveNegative Malignant tumor MSI-high adenocarcinoma Positive NegativeMalignant tumor Colloid adenocarcinoma Negative Negative Malignant tumorColloid adenocarcinoma Negative Negative Normal Normal mucosa NegativeNegative Normal Normal mucosa Negative Negative

The results in the table demonstrate that, in the healthy colonic mucosabiopsies, there is no labeling (10 negatives). The labeling is alsonegative in the adenoma (1/1). The labeling is positive in theepithelial cells of the colonic adenocarcinomas (+ in 8/11 patients).There is no labeling in the stroma.

3. Immunohistochemical Detection of Ezrin

Tissue-microarray slides were used to screen a large number of samples.These samples are colonic tissues spotted onto slides. For each patientwith a colonic adenocarcinoma, 3 samples were taken at the center of thetumor, 3 samples were taken at the invasion front and 3 samples weretaken in the healthy tissue. Table 13 shows the results of theimmunolabelings with the anti-Ezrin antibody; the level of labelingindicated is the maximum intensity over the 3 samples analyzed.

TABLE 13 Patient Tumor invasion identifier Tumor center front Healthytissue 55 + ++ 0 127 + + 0 329 + ++ + 475 + ++ + 544 + ++ + 726 + + +1203 ++ ++ + 1310 ++ +++ + 2003 + 0 + 2296 ++ ++ 0 2301 + ++ + 2377 + +0 3095 + + 0 3430 + + 0 3636 + + 0 3748 + + 0 3839 + ++ 0 3891 0 0 04054 + + 0 4322 + ++ 0 445 0 ++ + 4474 ++ ++ 0 4792 + + + 4958 ++ ++ +5101 + ++ + 5318 ++ +++ 0 5374 + + 0 5472 + 0 + 6340 ++ + 0 6353 ++ + 0

In a sampling of 30 patients, 25 exhibit overexpression of Ezrin in thetumor (tumor center or invasion front) compared with the adjacenthealthy tissue.

4. Immunohistochemical Detection of Aminoacylase 1

Tissue-microarray slides were used to screen a large number of samples.These samples are colonic tissues spotted onto slides. For each patientwith a colonic adenocarcinoma, 3 samples were taken at the center of thetumor, 3 samples were taken at the invasion front and 3 samples weretaken in the healthy tissue. Table 14 shows the results of theimmunolabelings with the anti-Aminoacylase antibody; the level oflabeling indicated is the maximum intensity over the 3 samples analyzed.

TABLE 14 Patient Tumor invasion identifier Tumor center front Healthytissue 55 ++ ++ 0 127 0 0 0 329 ++ ++ 0 475 ++ ++ + 544 + 0 0 726 0 0 01203 0 + 0 1310 0 + + 2003 ++ 0 0 2296 + + 0 2301 + + + 2377 + + +3095 + + 0 3430 + + + 3636 ++ + + 3748 ++ ++ 0 3839 ++ ++ + 3891 ++ ++ 04054 + ++ 0 4322 +++ +++ + 445 + ++ + 4474 + ++ + 4792 ++ ++ +4958 + + + 5101 + + ++ 5318 +++ ++ 0 5374 + + 0 5472 ++ ++ + 6340 ++++ + 6353 ++ ++ ++

In a sampling of 30 patients, 21 exhibited overexpression ofAminoacylase in the tumor (tumor center or invasion front) compared withthe adjacent healthy tissue.

5. Immunohistochemical Detection of 1-Plastin

Tissue-microarray slides were used to screen a large number of samples.These samples are colonic and rectal tissues spotted onto slides. Thecharacteristics of the patients (characteristics of the colonic tissuespots present on the colorectal cancer tissue-microarray), and also theresults of the immunolabelings with the anti-I-Plastin antibody, arereproduced in Table 15.

TABLE 15 Histology and genetic Labeling in the Labeling in the Diagnosischaracterization epithelial cells stroma Malignant colon tumor Conservedadenocarcinoma ++ Negative Malignant colon tumor Conservedadenocarcinoma ++ Negative Normal colon Normal mucosa + Negative Normalcolon Normal mucosa + Negative Normal colon Normal mucosa + NegativeBenign colon tumor Adenoma + Negative Malignant colon tumor Conservedadenocarcinoma + Negative Malignant colon tumor LOH adenocarcinoma ++Negative Malignant colon tumor LOH adenocarcinoma ++ Negative Normalcolon Normal mucosa + Negative Normal colon Normal mucosa + NegativeNormal colon Normal mucosa + Negative Malignant colon tumor LOHadenocarcinoma ++ Negative Malignant colon tumor LOH adenocarcinoma ++Negative Malignant colon tumor MSI-high adenocarcinoma + Negative Normalcolon Normal mucosa + Detached Normal colon Normal mucosa + DetachedMalignant colon tumor Colloid adenocarcinoma + Negative Normal colonNormal mucosa ++ Negative Normal colon Normal mucosa ++ Negative Normalrectum Normal rectal mucosa ++ Negative Normal rectum Normal rectalmucosa Nonspecific Negative Normal rectum Normal rectal mucosaNonspecific Negative Normal rectum Normal rectal mucosa NonspecificNegative Malignant rectal tumor LOH adenocarcinoma + Negative Malignantrectal tumor LOH adenocarcinoma ++ Negative Malignant rectal tumor LOHadenocarcinoma ++ Negative Malignant rectal tumor LOH adenocarcinoma ++Negative Benign rectal tumor Adenoma with low-grade dysplasia ++Negative Benign rectal tumor Adenoma with low-grade dysplasia ++Negative Benign rectal tumor Adenoma with low-grade dysplasia + NegativeBenign rectal tumor Adenoma with low-grade dysplasia + Negative Benignrectal tumor Adenoma with low-grade dysplasia + Negative Benign rectaltumor Adenoma with low-grade dysplasia + Negative Benign rectal tumorAdenoma with low-grade dysplasia ++ Negative Benign rectal tumor Adenomawith low-grade dysplasia ++ Negative Benign rectal tumor Adenoma withlow-grade dysplasia ++ Negative The results in the table demonstratethat: in the healthy colonic mucosa biopsies, the labeling is weak in 8samples (+) and 2 samples are ++. The labeling is also weak (+) in thecolonic adenoma (1/1). The labeling is strongly positive ++ in theepithelial cells of the colonic adenocarcinomas (++ in 6/9 patients and3 weak +, including the colonic colloid adenocarcinomas). There is nolabeling in the stroma; in the healthy rectal mucosa biopsies, labelingis present in the surface epithelium in a nonspecific manner (3/4) andat ++ level in one sample. The labeling is strongly positive ++ in therectal adenomas (5/9) or discreet + (4/9). The labeling is also strong++ in the epithelial cells of the rectal adenocarcinomas (++ in 3/4patients, 1 weak +). There is no labeling in the stroma.

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1. A method for the in vitro diagnosis of colorectal cancer comprising:measuring the level of expression of Ezrin in a blood, serum, or plasmasample from an individual using at least one anti-Ezrin monoclonalantibody directed against an epitope selected from the group consistingof SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4+SEQ ID No. 5,SEQ ID No. 6+SEQ ID No. 7 and SEQ ID No. 8; and diagnosing theindividual with colorectal cancer when the level of expression isincreased relative to a reference value indicative of the level ofexpression of Ezrin in an individual not having colorectal cancer. 2.The method as claimed in claim 1, comprising the use of at least oneanti-Ezrin monoclonal antibody directed against an epitope of SEQ ID No.1 and at least one other monoclonal antibody directed against an epitopeselected from the group consisting of SEQ ID No. 2, SEQ ID No. 4+SEQ IDNo. 5 and SEQ ID No. 6+SEQ ID No.
 7. 3. The method as claimed in claim2, comprising the use of at least one anti-Ezrin monoclonal antibodydirected against an epitope of SEQ ID No. 1 and at least one otherantibody directed against an epitope of SEQ ID No. 4+SEQ ID No.
 5. 4.The method as claimed in claim 1, comprising the use of at least onemonoclonal antibody directed against an epitope of SEQ ID No.
 2. 5. Themethod as claimed in claim 1, further comprising determining thepresence of at least one other tumor marker selected from the groupconsisting of Leukocyte Elastase Inhibitor, Aminoacylase 1, Liver FattyAcid-Binding Protein, Intestinal Fatty Acid-Binding Protein,Apolipoprotein AI, Apolipoprotein AII and I-Plastin.
 6. The method asclaimed in claim 1, further comprising determining the presence of atleast one other tumor marker selected from the group consisting ofBeta2-Microglobulin, Proteasome 20S, Galectin-3, L-Lactate DehydrogenaseChain B, Calreticulin, Regenerating Islet-Derived Protein 3 Alpha,Tumor-Associated Calcium Signal Transducer 1, Keratin type IICytoskeletal 8, Keratin type I Cytoskeletal 18, Keratin type ICytoskeletal 19, Epithelial-Cadherin, CEA, Villin, CA19-9, CA 242, CA50, CA 72-2, Testosterone, TIMP-1, Cripto-1, Intelectin-1, ProteinDisulfide Isomerase, Cytokeratin 20, Translationally-Controlled TumorProtein, (Pro)defensin-A5, methylated DNA in the blood, specificalterations in fecal DNA fragments, and fecal human hemoglobin.
 7. Themethod as claimed in claim 1, further comprising determining thepresence of at least one other tumor marker selected from the groupconsisting of Beta2-Microglobulin, Proteasome 20S, Galectin-3, L-LactateDehydrogenase Chain B, Calreticulin, Regenerating Islet-Derived Protein3 Alpha, Tumor-Associated Calcium Signal Transducer 1,Epithelial-Cadherin, CEA, CA19-9, Testosterone, TIMP-1, Intelectin-1,Protein Disulfide Isomerase, Cytokeratin 20, Translationally-ControlledTumor Protein, (Pro)defensin-A5, and fecal human hemoglobin.
 8. Themethod as claimed in claim 1, further comprising determining thepresence of Aminoacylase 1 marker.
 9. The method as claimed in claim 1,further comprising determining the presence of Beta 2-Microglobulin,Aminoacylase 1 and CEA markers.
 10. The method as claimed in claim 1,further comprising determining the presence of Beta 2-Microglobulin,Aminoacylase 1, Proteasome 20S, L-FABP, PAP, E-Cadherin and CEA markers.